WO2021218879A1 - Sars-cov-2中和抗体及其制备和应用 - Google Patents

Sars-cov-2中和抗体及其制备和应用 Download PDF

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WO2021218879A1
WO2021218879A1 PCT/CN2021/089748 CN2021089748W WO2021218879A1 WO 2021218879 A1 WO2021218879 A1 WO 2021218879A1 CN 2021089748 W CN2021089748 W CN 2021089748W WO 2021218879 A1 WO2021218879 A1 WO 2021218879A1
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antibody
sars
cov
cov2
antigen
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PCT/CN2021/089748
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French (fr)
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谢良志
孙春昀
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神州细胞工程有限公司
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    • 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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells

Definitions

  • the present invention relates to the technical field of cellular immunity, and provides a humanized antibody that can block the SARS-CoV-2 spike protein (S protein) from binding to the ACE2 receptor and efficiently neutralize SARS-CoV-2 virus-infected cells. It can be used to treat infectious diseases caused by SARS-CoV-2.
  • the present invention also provides a nucleic acid sequence encoding the antibody, a vector and a cell containing the nucleic acid sequence.
  • SARS-CoV-2 The spread of SARS-CoV-2 from person to person is similar to SARS-CoV (Severe Acute Respiratory Syndrome coronavirus) and MERS-CoV (Middle East Respiratory Syndrome). It is mainly spread by respiratory droplets and can also be spread through contact.
  • the main source of infection of SARS-CoV-2 is COVID-19 patients, and those with asymptomatic infection may also become the source of infection.
  • Relevant studies have shown that the basic infection number (R0 value) of SARS-CoV-2 is between 2.24 and 3.58, suggesting that it has a strong transmission capacity [1] .
  • the population is generally susceptible to SARS-CoV-2, with an incubation period of 1-14 days, mostly 3-7 days.
  • the New Coronavirus Pneumonia Diagnosis and Treatment Plan (Trial Seventh Edition)" divides COVID-19 into mild, normal, severe, and critical.
  • the clinical symptoms of COVID-19 are mainly fever, dry cough, and fatigue.
  • a few patients are accompanied by nasal congestion, runny nose, sore throat, myalgia, and diarrhea.
  • Mild patients only manifested as low-grade fever, mild fatigue, etc., without pneumonia.
  • Critically ill patients usually have difficulty breathing and/or hypoxemia one week after the onset of onset. In severe cases, they can quickly progress to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis and coagulation dysfunction, and Multiple organ failure, etc. [2,3] .
  • SARS-CoV-2 and SARS-CoV share a common host cell receptor protein, angiotensin converting enzyme 2 (ACE2) [4] .
  • ACE2 angiotensin converting enzyme 2
  • S protein trimeric spike protein
  • the trimeric spike protein (S protein) of the virus binds to the ACE2 receptor and is cleaved by the host protease into the S1 polypeptide containing the receptor binding domain (RBD) and the S2 polypeptide responsible for mediating the fusion of the virus with the cell membrane. Then invade the body [5] . Therefore, finding and preparing effective antibodies to prevent the SARS-CoV-2 RBD protein from binding to the ACE2 receptor, thereby inhibiting the virus from infecting cells, has become the top priority for the prevention and treatment of SARS-CoV-2 virus infection.
  • RBD receptor binding domain
  • the humanized monoclonal antibody of the invention can effectively neutralize SARS-CoV-2 virus, and can be used as a specific antibody medicine to prevent and treat acute respiratory infections caused by SARS-CoV-2 virus.
  • the present invention provides an isolated binding antibody or antigen-binding fragment thereof that blocks the SARS-CoV-2 spike protein and ACE2 receptor, which comprises
  • the heavy chain variable region whose heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 domains respectively comprise SEQ ID NOs: 13, 14 and 15, and/or
  • the light chain variable region, the light chain CDR1, light chain CDR2 and light chain CDR3 domains respectively comprise SEQ ID NOs: 10, 11 and 12
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain variable region whose sequence includes SEQ ID NO: 22 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it;
  • the light chain variable region the sequence of which comprises SEQ ID NO: 23 or has at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it.
  • the antibody or antigen-binding fragment thereof is a humanized antibody, chimeric or murine antibody.
  • the antibody or antigen-binding fragment thereof has an antibody constant region of IgG, IgM, IgA subtype, preferably, IgG1, IgG2, or IgG4 subtype antibody; more preferably, because of its Fc region IgG1, IgG2 or IgG4 subtype antibodies with altered amino acid sequence and/or glycosylation form of Fc receptor and/or C1q complement binding function.
  • the antibody or antigen-binding fragment thereof in one embodiment, the antibody or antigen-binding fragment thereof,
  • the average K D of its binding affinity to SARS-CoV-2 RBD is 11.7E-11 to 1.3E-11M, preferably 5.9E-11 to 2.6E-11M, more preferably 3.9E-11M; and/or
  • the average K D of its binding affinity to SARS-CoV S1 is 11.1E-10 to 1.2E-10M, preferably 5.6E-10 to 2.5E-10M, more preferably 3.7E-10M.
  • the epitope of the antibody or antigen-binding fragment thereof is N439 and T500 of the SARS-CoV-2 virus spike protein.
  • the antibody or antigen-binding fragment thereof further comprises:
  • the heavy chain constant region preferably, its sequence comprises SEQ ID NO: 24 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it; and/or
  • the light chain constant region preferably, its sequence comprises SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it.
  • the average in vivo exposure C max and AUC last are 108.96 ⁇ g/mL and 9922.11 h ⁇ g/mL, respectively, and the average half-life is t 1/2 It was 286.16h, and the clearance rate Cl was 0.30mL/h/kg.
  • the antibody is expressed by HEK-293 knocked out of fut8 gene.
  • the antibody further comprises:
  • the heavy chain constant region preferably, its sequence comprises SEQ ID NO: 45 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it; and/or
  • the light chain constant region preferably, its sequence comprises SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it.
  • CD32a and CD32b There is almost no combination with CD32a and CD32b;
  • the average in vivo exposure C max and AUC last are 115.04 ⁇ g/mL and 11159.82 h ⁇ g/mL, respectively, and the average half-life is t 1/2 It is 320.00h, and the clearance rate Cl is 0.23mL/h/kg.
  • the antibody is expressed by HEK-293 knocked out of fut8 gene.
  • the binding ability to CD16a is stronger than that of CoV2-HB27 antibody of IgG1 subtype
  • the antibody further comprises
  • the heavy chain constant region preferably, its sequence comprises SEQ ID NO: 49 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it; and/or
  • the light chain constant region preferably, its sequence comprises SEQ ID NO: 25 or has at least 90%, 92%, 95%, 98% or 99% sequence identity with it.
  • the antibody is expressed by HEK-293 knocked out of fut8 gene.
  • the binding ability to CD16a is stronger than that of CoV2-HB27 antibody of IgG1 subtype
  • the binding capacity to CD64 is stronger than that of CoV2-HB27 antibody of IgG1 subtype
  • CoV2-HB27 antibody that mediates ADCC stronger than IgG1 subtype
  • the antibody or antigen-binding fragment thereof is a monoclonal antibody.
  • the antigen-binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecule or single-domain antibody;
  • the antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.
  • the present invention provides a modified antibody-drug molecule, which comprises the antibody of the present invention or its antigen-binding fragment and small molecules or biological macromolecules covalently or non-covalently linked thereto, preferably via a linker connect.
  • the present invention provides a nucleic acid, which encodes the antibody or antigen-binding fragment thereof according to the present invention, which is mRNA and/or DNA.
  • the nucleic acid comprises
  • the present invention provides an expression vector comprising the nucleic acid according to the present invention.
  • the present invention provides a host cell comprising the nucleic acid according to the present invention or the expression vector according to the present invention.
  • the present invention provides a method for producing the antibody or antigen-binding fragment thereof according to the present invention, which comprises culturing the host cell according to the present invention under conditions suitable for antibody expression, and from The expressed antibody is recovered from the culture medium.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising
  • a pharmaceutically acceptable carrier optionally
  • One or more other therapeutic agents are selected from antiviral drugs or inflammatory factor inhibitors, small molecule chemical drugs of other mechanisms; preferably, the antiviral drugs are selected from including but not limited to type I interferon Drugs, antibodies, protease inhibitors, RNA-dependent RNA polymerase (RdRP) inhibitors, and host-targeted antiviral drugs.
  • antiviral drugs are selected from including but not limited to type I interferon Drugs, antibodies, protease inhibitors, RNA-dependent RNA polymerase (RdRP) inhibitors, and host-targeted antiviral drugs.
  • the antibody or antigen-binding fragment thereof according to the present invention is used to prevent and treat diseases caused by SARS-CoV-2 infection.
  • the antibody or antigen-binding fragment thereof according to the present invention is used to prepare a medicine for preventing and treating diseases caused by SARS-CoV-2 infection.
  • the present invention provides a pharmaceutical combination comprising
  • the antibody or antigen-binding fragment thereof according to the present invention the modified antibody-drug molecule according to the present invention, the nucleic acid according to the present invention, the expression vector according to the present invention, the drug according to the present invention Composition; and one or more additional therapeutic agents.
  • the present invention provides a kit comprising
  • the antibody or antigen-binding fragment thereof according to the present invention, the modified antibody-drug molecule according to the present invention, the nucleic acid according to the present invention, the expression vector according to the present invention, the drug according to the present invention The composition; preferably, it further comprises a device for administration.
  • the present invention provides a method for preventing and treating diseases caused by SARS-CoV-2 infection, which comprises administering to a subject the antibody or antigen-binding fragment thereof according to the present invention, as described in the present invention.
  • the present invention provides an isolated binding antibody or antigen-binding fragment thereof that blocks the SARS-CoV-2 spike protein and ACE2 receptor, and its binding epitope is N439 of the SARS-CoV-2 spike protein And T500.
  • the present invention provides a SARS-CoV-2 spike protein binding epitope, which is N439 and T500 of SARS-CoV-2 spike protein.
  • Figure 1 Screening of monoclonal phage binding to SARS-CoV-2 protein.
  • Figure 2 The binding ability of murine antibodies with SARS-CoV-2 S1 and ECD proteins.
  • Figure 3 Flow cytometric detection of the binding of mouse antibodies to SARS-CoV-2 S1 protein.
  • FIG. 4 Murine antibody competes for the binding of ACE2 to SARS-CoV-2 RBD and S1 protein.
  • FIG. 5 Murine antibody neutralizes SARS-CoV-2 pseudovirus.
  • Figure 6 The binding ability of humanized antibodies with SARS-CoV-2 RBD, S1 and ECD proteins.
  • Figure 7 Humanized antibodies compete for the binding of ACE2 protein to SARS-CoV-2 S1 protein.
  • Figure 8 The affinity detection of humanized antibody with SARS-CoV-2 RBD protein and S1 protein.
  • Figure 9 Humanized antibody neutralizes SARS-CoV-2 pseudovirus.
  • FIG. 10 Epitope diagram of CoV2-mhB27 epitope analysis (A) and ELISA test results (B). SARS-CoV-2RBD in A is represented by a white surface model, all designed mutation sites are represented by light gray, and the identified highly significant epitope of mhB27 is represented by black.
  • FIG. 11 Binding of CoV2-HB27 antibodies with different Fc functional forms to CD16a.
  • Figure 12 Binding of CoV2-HB27 antibodies to CD32 in different Fc functional forms.
  • Figure 13 Binding of CoV2-HB27 antibodies with different Fc functional forms to CD64.
  • FIG. 14 Binding of CoV2-HB27 antibodies with different Fc functional forms to C1q.
  • Figure 15 Different Fc functional forms of CoV2-HB27 antibody on ADCC mediated by target cells expressing SARS-CoV-2 S protein.
  • FIG. 16 ADCP mediated by different Fc functional forms of CoV2-HB27 antibodies on target cells expressing SARS-CoV-2 S protein.
  • Figure 17 CDC mediated by different Fc functional forms of CoV2-HB27 antibodies on target cells expressing SARS-CoV-2 S protein.
  • antibody means an immunoglobulin molecule, and refers to any form of antibody that exhibits the desired biological activity. Including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (such as bispecific antibodies), and even antibody fragments.
  • the full-length antibody structure preferably comprises 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains connected to each other by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, its structure also includes other derivative forms.
  • variable region refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen.
  • the variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures and can be further subdivided into hypervariable regions interspersed in more conservative regions (called framework regions (FR)) (Called the complementarity determining region (CDR)).
  • CDR complementarity determining region
  • CDR1, CDR2, and CDR3 refers to the amino acid residues of the variable region of an antibody, the presence of which is necessary for antigen binding.
  • Each variable region usually has 3 CDR regions identified as CDR1, CDR2, and CDR3.
  • Each complementarity determining region may contain amino acid residues from the “complementarity determining region” defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). .1991)) and/or those residues from the "hypervariable loop" (Chothia and Lesk; J Mol Biol 196:901-917 (1987)).
  • framework or "FR” residues are those variable region residues other than the CDR residues as defined herein.
  • Each heavy chain variable region and light chain variable region usually contains 3 CDRs and up to 4 FRs, and the CDRs and FRs are arranged in the following order, for example, from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDR complementarity determining region
  • FR framework region
  • constant region refers to such amino acid sequences on the light chain and heavy chain of an antibody that do not directly participate in the binding of the antibody to the antigen, but exhibit a variety of effector functions, such as antibody-dependent cytotoxicity.
  • complete antibodies can be classified into five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, among which IgG and IgA can be further divided into subclasses (isotypes), such as IgG1, IgG2 , IgG3, IgG4, IgA1 and IgA2.
  • the heavy chains of the five types of antibodies are classified into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains, respectively.
  • the amino acid sequence of the constant region of its light chain the light chain of an antibody can be classified into ⁇ and ⁇ . .
  • an "antigen-binding fragment of an antibody” comprises a part of a complete antibody molecule that retains at least some of the binding specificity of the parent antibody, and usually includes at least part of the antigen-binding region or variable region (for example, one or more CDRs) of the parent antibody.
  • antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single-chain antibody molecules (e.g., scFv, di-scFv, or tri-scFv , Diabody or scFab), single domain antibody.
  • antibody fragment is a non-complete antibody molecule that retains at least some biological properties of the parent antibody, and examples thereof include, but are not limited to, Fc fragments in addition to those described in the above-mentioned "antigen-binding fragments".
  • modified drug molecule refers to an antibody or fragment thereof, such as an antigen-binding fragment that forms a covalent or non-covalent link with another molecule or forms a recombinant multi-target fusion drug, and the other molecule is selected from a small molecule compound or a biological Macromolecule.
  • chimeric antibody refers to an antibody in which a part of the heavy chain and/or light chain is derived from a specific source or species, and the remaining part is derived from a different source or species.
  • Humanized antibodies are a subset of “chimeric antibodies.”
  • humanized antibody or “humanized antigen-binding fragment” is defined herein as an antibody or antibody fragment: (i) derived from a non-human source (for example, a transgenic mouse carrying a heterologous immune system) And antibodies based on human germline sequences; or (ii) chimeric antibodies in which the variable region is of non-human origin and the constant region is of human origin; or (iii) CDR-grafted, in which the CDR of the variable region is derived from a non-human source, and One or more framework regions of the variable region are of human origin, and the constant region (if any) is of human origin.
  • the purpose of "humanization” is to eliminate the immunogenicity of non-human source antibodies in the human body, while retaining the greatest possible affinity.
  • a “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, that is, the population comprising a single antibody is identical except for possible mutations (such as natural mutations) that may be present in very small amounts. Therefore, the term “monoclonal” indicates the nature of the antibody, that is, it is not a mixture of unrelated antibodies. In contrast to polyclonal antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single set of determinants on the antigen. In addition to their specificity, the advantage of monoclonal antibody preparations is that they are generally not contaminated by other antibodies. The term “monoclonal” should not be understood as requiring the production of the antibody by any specific method. The term monoclonal antibody specifically includes chimeric antibodies, humanized antibodies and human antibodies.
  • the antibody "specifically binds" to an antigen of interest, such as a virus-associated antigen protein (herein, spike protein S), that is, binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent, and targets those expressing the antigen.
  • an antigen of interest such as a virus-associated antigen protein (herein, spike protein S)
  • spike protein S virus-associated antigen protein
  • Viruses or cells and have no significant cross-reactivity with other proteins or with proteins other than homologs and variants (such as mutant forms, splice variants, or proteolytically truncated forms) of the antigen target mentioned above No significant cross-reaction.
  • binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise specified, "binding affinity” as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). "KD”, "association rate constant kon” and “dissociation rate constant koff” are generally used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), that is, how tightly a ligand binds to a specific protein. Binding affinity is affected by interactions between non-covalent molecules, such as hydrogen bonds, electrostatic interactions, hydrophobicity between two molecules and van der Waals forces. In addition, the binding affinity between the ligand and its target molecule may be affected by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA described herein.
  • epitope includes any protein determinant capable of specifically binding to an antibody or T cell receptor.
  • Epitope determinants usually consist of chemically active surface groups of molecules (such as amino acids or sugar side chains, or combinations thereof), and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • Isolated antibodies are antibodies that have been identified and isolated from cells that naturally express the antibody. Isolated antibodies include in situ antibodies in recombinant cells as well as antibodies that are usually prepared through at least one purification step.
  • sequence identity between two polypeptide or nucleic acid sequences means the percentage of the number of residues that are identical between the sequences to the total number of residues.
  • sequences being compared are aligned in a way that produces the largest match between the sequences, and the gaps in the alignment (if any) are resolved by a specific algorithm.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package, including GAP, BLASTP, BLASTN and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410) .
  • the above procedures are publicly available from the International Center for Biotechnology Information (NCBI) and other sources.
  • NCBI International Center for Biotechnology Information
  • Smith Waterman algorithm can also be used to determine identity.
  • receptor is a biochemical concept that refers to a class of molecules that can transmit extracellular signals and produce specific effects in cells. The effect may only last for a short period of time, such as changing cell metabolism or cell movement. It may also be a long-term effect, such as up-regulating or down-regulating the expression of a certain gene or genes.
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody. Natural sequence human FcR is preferred, and receptors ( ⁇ receptors) that bind to IgG antibodies are preferred, which include Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII subtypes, and variants of these receptors. Other FcRs are included in the term “FcR”.
  • the term also includes the neonatal receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al., Journal of Immunology 117:587 (1976) and Kim et al., Journal of Immunology 24:249 (1994)).
  • FcRn neonatal Fc receptor
  • the neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG antibodies in the body. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and increasing its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in the blood circulation.
  • effector function refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, "antibody-dependent cell-mediated cytotoxicity” (ADCC), antibody-dependent cellular phagocytosis (ADCP), Cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors), and B cell activation.
  • effector cells refers to leukocytes that express one or more FcRs and perform effector functions.
  • the effector cell at least expresses FcyRIII and performs ADCC effector function.
  • human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells
  • neutrophils effector cells can be isolated from natural sources, for example, blood. Effector cells are usually lymphocytes associated with the effector stage and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells) or secrete antibodies (differentiated B cells) .
  • Immune cells include cells that have hematopoietic origin and play a role in immune responses. Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as NK cells, neutrophils, and macrophages.
  • Ig on the Fc ⁇ receptor enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen, and then kill the target cell using, for example, a cytotoxin.
  • an in vitro ADCC assay can be performed, such as the in vitro ADCC assay described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), and the method described in the Examples of this application .
  • Useful effector cells for such assays include PBMC and NK cells.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement.
  • the activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass), wherein the antibody binds to its corresponding antigen.
  • C1q the first component of the complement system
  • CDC assays can be performed, such as the CDC assays described in Gazzano-Santoro et al., J. Immunol Methods 202:163 (1996), such as the methods described in the examples of this application, such as The method described in U.S. Patent No. 6,194,551B1 and WO1999/51642, in which polypeptide variants with altered Fc region amino acid sequence (polypeptides with variant Fc region) and polypeptide variants with enhanced or reduced C1q binding are described .
  • ADCP antibody-dependent cellular phagocytosis
  • amino acid sequence and nucleotide sequence of the antibody of the present invention as well as Fc functional modification.
  • the present invention first uses recombinant SARS-CoV RBD protein to immunize mice, and then obtains a scFv antibody clone that binds to SARS-CoV-2 RBD protein through screening of the phage antibody library. After that, the nucleotide sequences encoding the heavy chain and light chain variable regions of the scFv antibody were spliced with the nucleotide sequences encoding the mouse IgG1 heavy chain constant region and the mouse kappa light chain constant region, respectively, and inserted into the transient Transform the expression vector for culture expression. The protein A purification column was used for purification to obtain high-purity mouse antibodies.
  • the classical CDR transplantation method is used to humanize the mouse antibody [8,9] .
  • the similarity with the murine light chain and heavy chain variable regions is more than 50%
  • the framework regions of the light chain and heavy chain variable regions are similar to the amino acid sequences of the framework regions of the light chain and heavy chain variable regions of the antibody to be modified
  • An antibody with a sex ratio of more than 50% is used as a humanized template, and the three CDR sequences of the light chain or heavy chain of the mouse antibody are replaced in the corresponding CDR amino acid sequence in the humanized template.
  • human-source templates for light and heavy chain variable region transplantation were selected.
  • the 3 CDR sequences of the light chain and the heavy chain of the murine antibody were transplanted into the corresponding human template. Since the key points of the mouse-derived framework region are essential to support the activity of the CDR, the key points were backmutated to the sequence of the murine antibody.
  • the light chain/heavy chain signal peptide sequence, the back-mutated humanized antibody light chain/heavy chain variable region sequence, and the human IgG4 heavy chain constant region/human kappa light chain constant region sequence were spliced in sequence to obtain the humanization
  • the amino acid sequence and nucleotide sequence of the antibody CoV2-HB27.
  • the present invention further modified the Fc function of CoV2-HB27.
  • They are: 1) genetically engineered heavy chain IgG1 constant region to obtain a humanized antibody CoV2-HB27-Fd6-IgG1 with reduced Fc function IgG1 subtype; 2) Defucoid expressed in mammalian cells knocked out with Fut8 gene Glycosylated IgG1 subtype CoV2-HB27-Ae0-IgG1 antibody; and 3) genetically engineered heavy chain IgG1 constant region to obtain an enhanced Fc function IgG1 subtype humanized antibody CoV2-HB27-Fe4-IgG1.
  • the invention also relates to nucleic acid molecules encoding the antibodies of the invention or parts thereof. Some example sequences of these nucleic acid molecules are shown in the sequence listing.
  • the nucleic acid molecule of the present invention is not limited to the sequence disclosed herein, but also includes variants and other nucleic acid forms corresponding thereto, such as mRNA, cDNA and variants thereof.
  • the variants of the present invention can be described with reference to their physical characteristics in hybridization. Those skilled in the art will recognize that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements and their equivalents or homologs. It will also be recognized that hybridization can occur with less than 100% complementarity. However, considering the appropriate selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural correlation with specific probes.
  • the invention also provides a recombinant construct comprising one or more nucleotide sequences of the invention.
  • the recombinant construct of the present invention can be used with a vector, such as a plasmid, phagemid, phage or viral vector, into which the nucleic acid molecule encoding the antibody of the present invention is inserted.
  • the antibodies provided herein can be prepared by recombinantly expressing nucleotide sequences encoding light and heavy chains or parts thereof in a host cell.
  • one or more recombinant expression vectors carrying the nucleotide sequence encoding the light chain and/or the heavy chain or part thereof can be used to transfect the host cell so that the light chain and the heavy chain are in the Expressed in host cells.
  • Standard recombinant DNA methodology is used to prepare and/or obtain nucleic acids encoding heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as Sambrook, Fritsch and Maniatis (eds.
  • the nucleotide sequence encoding the variable region of the heavy chain and/or light chain can be converted into, for example, a nucleotide sequence encoding a full-length antibody chain, Fab fragment or scFv: for example, the variable region encoding the light chain can be
  • the DNA fragment of the region or heavy chain variable region is operably linked (so that the amino acid sequences encoded by the two DNA fragments are in frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker.
  • the sequences of human heavy and light chain constant regions are known in the art (see, for example, Kabat, EA, el. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), DNA fragments including these regions can be obtained by standard PCR amplification.
  • the nucleotide sequence encoding the desired antibody can be inserted into an expression vector, and then the expression vector can be transfected into a suitable host cell.
  • suitable host cells are prokaryotic cells and eukaryotic cells. Examples of prokaryotic host cells are bacteria, and examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of the expression vector including the selection regulatory sequence is affected by many factors, such as the choice of host cell, the expression level of the desired protein, and whether the expression is constitutive or inducible.
  • the antibody of the present invention can be recovered and purified from recombinant cell culture by known methods, including but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion Or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • High performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), such as Chapters 1, 4, 6, 8, 9, and 10, each by reference The full text is included in this article.
  • the antibodies of the present invention include natural purified products, products of chemical synthesis methods, and products produced by recombinant technology from prokaryotic and eukaryotic hosts.
  • the eukaryotic hosts include, for example, yeast, higher plants, insects and mammalian cells.
  • the antibodies of the invention can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook above, sections 17.37-17.42; Ausubel above, chapters 10, 12, 13, 16, 18 and 20.
  • an embodiment of the present invention is also a host cell comprising the vector or nucleic acid molecule, wherein the host cell may be a higher eukaryotic host cell such as mammalian and insect cells, a lower eukaryotic host cell such as yeast cell, and It may be a prokaryotic cell such as a bacterial cell.
  • the host cell may be a higher eukaryotic host cell such as mammalian and insect cells, a lower eukaryotic host cell such as yeast cell, and It may be a prokaryotic cell such as a bacterial cell.
  • the ELISA test showed that the obtained mouse antibody CoV2-mhB27 has a good binding with the transiently expressing SARS-CoV-2 S1 protein HF-nCoV-SPIKE-8D3 cells, and its binding EC 50 is 2.77 ⁇ g/mL; it can effectively compete with ACE2
  • the binding of the protein to RBD and S1 protein has a competitive EC 50 of 44.0ng/mL and 24.0ng/mL, respectively; it can effectively neutralize the SARS-CoV-2 pseudovirus in a concentration-dependent manner, with a neutralization IC 50 of 8.7ng /mL.
  • the humanized antibody CoV2-HB27 has good binding with SARS-CoV-2 RBD, S1 and ECD proteins.
  • the binding exhibits an "S" curve growth, and the binding EC 50 is 22.2ng/mL, 183.3ng/mL and respectively. 972.4ng / mL; can be effectively suppressed with the S1 binding protein 2 SARS-CoV-ACE2 protein, inhibition EC 50 of 178.6ng / mL.
  • CoV2-HB27 has high affinity with SARS-CoV-2 RBD and S1 protein.
  • CoV2-HB27 can effectively neutralize SARS-CoV2 pseudovirus, and concentration-dependent manner, and the IC 50 of 5.9ng / mL.
  • the defucosylated Ae0-IgG1 form antibody and the Fc-enhanced Fe4-IgG1 form antibody have similar binding capacity to CD16a, and are stronger than the IgG1 subtype CoV2-HB27 antibody ;
  • the Fd6-IgG1 form of antibody that reduces Fc function only weakly binds to CD16a.
  • the Fe4-IgG1 form antibody with enhanced Fc function has the strongest binding ability to CD32a and CD32b, which is better than the defucosylated Ae0-IgG1 form antibody and IgG1 subtype CoV2 -HB27 antibody; Fd6-IgG1 form antibody which reduces Fc function has almost no binding to CD32a and CD32b.
  • the Fe4-IgG1 form antibody with enhanced Fc function has the strongest binding ability to CD64, which is better than the defucosylated Ae0-IgG1 form antibody and the IgG1 subtype CoV2-HB27 Antibody; Fd6-IgG1 form of antibody that reduces Fc function only weakly binds to CD64.
  • the Fe4-IgG1 form antibody with enhanced Fc function has the strongest binding ability to C1q, which is better than the defucosylated Ae0-IgG1 form antibody and IgG1 subtype CoV2-HB27 Antibody; Fd6-IgG1 form of antibody that reduces Fc function only weakly binds to C1q.
  • CoV2-HB27 antibodies have no CDC effect on target cells expressing SARS-CoV-2S protein.
  • the antibody of the present invention can be used to treat, prevent or detect diseases caused by SARS-CoV-2 virus, such as acute respiratory infectious diseases caused by SARS-CoV-2 virus.
  • One or more of the antibody, antigen-binding fragment, modified antibody-drug molecule, nucleic acid, carrier and at least one other chemical agent of the present invention can be prepared into a pharmaceutical composition, which includes the above-mentioned active ingredients and one or more A pharmaceutically acceptable carrier, diluent or excipient; optionally, one or more other therapeutic agents may also be included.
  • the invention also relates to a pharmaceutical package and a kit comprising one or more containers containing the above-mentioned pharmaceutical composition of the invention.
  • a pharmaceutical package and a kit comprising one or more containers containing the above-mentioned pharmaceutical composition of the invention.
  • Related to this type of container may be a reminder in the form prescribed by a government agency that regulates the production, use, or sale of drugs or biological products, which reflects that the product is approved for human administration by the agency that produces, uses, or sells the product.
  • the pharmaceutical composition of the present invention can be prepared in a manner known in the art, for example, by conventional mixing, dissolving, granulating, grinding, emulsifying, wrapping, embedding or freeze-drying methods.
  • the pharmaceutical composition containing the compound of the present invention formulated in an acceptable carrier After the pharmaceutical composition containing the compound of the present invention formulated in an acceptable carrier has been prepared, they can be placed in a suitable container and labeled for the treatment of the indicated condition.
  • suitable container and labeled for the treatment of the indicated condition.
  • labels would include the amount, frequency, and method of administration.
  • composition comprising the antibody of the present invention is also combined with one or more other therapeutic agents, wherein the resulting combination does not cause unacceptable adverse effects.
  • Example 1 Screening of murine antibodies that bind to SARS-CoV-2 RBD using phage antibody display library
  • the recombinant SARS-CoV-2-RBD-mFc protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.40592-V05H, SEQ ID NO:1, the same below) was used to immunize mice.
  • the specific method is: mixing recombinant SARS-CoV-2-RBD-mFc protein with Freund's adjuvant, using the mixture for secondary immunization, each immunization dose is 50 ⁇ g, subcutaneous injection, and the immunization interval is 2 weeks. Seven days after the second immunization, blood was collected from the intracanthal venous plexus of the orbit.
  • Coated recombinant SARS-CoV-2-RBD-his protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.40592-V08B), and ELISA method was used to detect the serum titer of mice after immunization.
  • the titer of the second immune serum was diluted 16000 times to reach 2.228. After 10 days, the mice were sacrificed, and the spleen tissues of the mice were frozen and stored in liquid nitrogen.
  • TriPure Isolation Reagent kit (source: Roche, Cat. No. 11 667 165 001) to extract RNA from mouse spleen tissue, and use reverse transcription kit TriPure Isolation Reagent (source: Invitrogen Cat. No. 18080-051) CDNA is obtained after reverse transcription.
  • the overlap extension splicing PCR method was used to splice the nucleotide sequence encoding the mouse antibody light chain and heavy chain variable region sequence into a scFv encoding Nucleotide sequence, light and heavy chain variable regions through linker:
  • the solid phase screening method was used to screen the phages that bind to the SARS-CoV-2 RBD protein.
  • SARS-CoV-2-RBD-mFc protein at a concentration of 10 ⁇ g/mL was coated on a 96-well plate with 100 ⁇ L per well and coated overnight at 4°C. The plate was washed the next day and blocked for 1 hour at room temperature, then added to the phage library and incubated at 37°C for 2 hours.
  • Monoclonal phages were selected from the enriched library for expression, and their binding to SARS-CoV-2 S and RBD proteins was detected by ELISA.
  • the SARS-CoV-2-S1-his protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., the same below)
  • the SARS-CoV-2-RBD-mFc protein and the negative control CD155(D1)- mFc (source: Shenzhou Cell Engineering Co., Ltd.) proteins were coated on 96-well plates, 100 ⁇ L per well, and coated overnight at 4°C. The plate was washed the next day and blocked at room temperature for 2 hours.
  • the plate was washed to remove unbound proteins, and then a 10-fold diluted phage monoclonal was added to incubate. The plate was washed to remove unbound phage, and M13/HRP was added to incubate and the plate was washed repeatedly. Add the substrate color solution for color development, and read the OD 450 by the microplate reader after termination. Take the detected protein as the abscissa and OD 450 as the ordinate, analyze and graph with GraphPad Prism software.
  • the nucleotide sequence of the heavy chain variable region of the CoV2-mB27scFv antibody was amplified by PCR, and inserted into the heavy chain signal peptide (SEQ ID NO: 40) and human IgG1 constant region (SEQ ID NO: 6) by the In-fusion method.
  • the expression vector of the human-mouse chimeric antibody CoV2-mhB27 heavy chain was obtained from the pSE vector (source: Shenzhou Cell Engineering Co., Ltd., the same below) digested with ScaI+Nhe I (source: Fermentas).
  • the nucleotide sequence of the light chain variable region of CoV2-mB27scFv was amplified by PCR and inserted into the light chain signal peptide (SEQ ID NO: 41) and human kappa constant region (SEQ ID NO: 7) by the In-fusion method.
  • the expression vector of human-mouse chimeric CoV2-mhB27 light chain (SEQ ID NO: 37) was obtained from pSE vector digested with ScaI+BsiWI (source: Fermentas, the same below).
  • Amplification variable region primers :
  • the culture solution was centrifuged at 4000 rpm for 25 min, the supernatant was collected, and 1/5 of the supernatant volume was added to stock buffer (source: Shenzhou Cell Engineering Co., Ltd.). Equilibrate the protein A chromatography column (source: Shenzhou Cell Engineering Co., Ltd., the same below) with PBS for 5-10 times the column volume, add the filtered culture supernatant to the chromatography column, and equilibrate again for 5-10 times the column volume, The sample was eluted with sodium acetate buffer (source: Shenzhou Cell Engineering Co., Ltd.). After the sample is eluted, it is neutralized to neutral with Tris buffer for later use.
  • stock buffer source: Shenzhou Cell Engineering Co., Ltd.
  • SARS-CoV-2 S1 protein 1000ng/mL, 333.3ng/mL, 111.1ng/mL, 37.0ng/mL, 12.4ng/mL, 4.12ng/mL and 1.37ng/mL
  • SARS-CoV -2ECD protein 9000ng/mL, 3000ng/mL, 1000ng/mL, 333.3ng/mL, 111.1ng/mL, 37.0ng/mL, 12.4ng/mL and 4.12ng/mL, source: Beijing Yiqiao Shenzhou Technology Co., Ltd. , The same below
  • the plate was washed the next day and blocked at room temperature for 1 hour, then 100 ⁇ L of 1 ⁇ g/mL murine antibody CoV2-mhB27 was added and incubated for 1 hour, then the plate was washed to remove unbound antibody, and 0.25 ⁇ g/mL Goat anti-human IgG Fc/HRP (source: KPL) was added. Company, the same below) Wash the plate repeatedly after incubation, add substrate color developing solution for color development, and detect OD 450 after termination.
  • CoV2-mhB27 antibody to SARS-CoV2 S1 binding protein and ECD protein showed an "S" curve growth, which binds EC 50 respectively 116.4ng / mL and 306.6ng / mL.
  • This example further verified the binding ability of the murine antibody with the transiently expressing SARS-CoV-2 S1 protein HF-nCoV-SPIKE-8D3 cell by flow cytometry. Take HF-nCoV-SPIKE-8D3 cells (source: Shenzhou Cell Engineering Co., Ltd.) in the logarithmic growth phase and place them in a flow tube, 5 ⁇ 10 5 cells/tube.
  • mice antibody CoV2-mhB27 33.3 ⁇ g/mL, 16.7 ⁇ g/mL, 8.3 ⁇ g/mL, 4.2 ⁇ g/mL, 2.1 ⁇ g/mL, 1.04 ⁇ g/mL and 0.52 ⁇ g/mL
  • H7N9 -R1 is a negative control antibody
  • the PBS washing solution was used to wash, and the unbound antibody was removed by centrifugation.
  • FITC-labeled Goat anti-Human IgG Fc secondary antibody source: KPL company
  • the cells were resuspended in 200 ⁇ L PBS, filtered with 400 meshes, and tested on a flow cytometer.
  • the results shown in Figure 3 CoV2-mhB27 antibody HF-nCoV-SPIKE-8D3 cells better binding, EC 50 which binds to 2.77 ⁇ g / mL, negative control without binding.
  • the SARS-CoV-2 RBD or S1 protein at a concentration of 1 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 4°C. The plate was washed the next day and blocked at room temperature for 1 hour, then 100 ⁇ L of 0.08 ⁇ g/mL ACE2 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., the same below) and different concentrations of CoV2-mhB27 antibody (2500ng/mL, 625ng/mL, 156.3ng/mL, 39.1ng/mL, 9.8ng/mL and 2.4ng/mL) were incubated together.
  • Inhibition rate PI% (OD blank- OD sample ) / OD blank ⁇ 100%, where OD blank represents the OD value of the normal coating with only ACE2 and no antibody group, and OD sample represents the detection of normal coating with ACE2 and antibody at the same time Group OD value.
  • the results are shown in Figure 4.
  • the ACE2 protein can bind to the RBD and S1 protein of the coated SARS-CoV-2, and the CoV2-mhB27 antibody can effectively compete for the binding of the ACE2 protein to the RBD and S1 protein.
  • the competition EC 50 is 44.0, respectively. ng/mL and 24.0ng/mL.
  • Lennti-X 293 (source: Clontech) was used to package pseudovirus expressing the full-length SARS-CoV-2 S protein.
  • a total of 62 ⁇ g of PSD, pWPXL-Luc and pCMV3-SARS-CoV-2-S plasmid (source: Shenzhou Cell Engineering Co., Ltd.) were mixed in a ratio of 3:4:2, and 72 ⁇ L of Sinofection TF02 (source: Beijing) Yiqiao Shenzhou Technology Co., Ltd.) transfection reagent. After mixing, let it stand at room temperature for 10 minutes, and then add it to Lenti-X 293 cells. Place the cell plate in a 37°C, 5% CO 2 incubator for 6 hours and then change the medium. After culturing for 48 hours, the supernatant was collected, and the cell debris was removed by filtration with a 0.45 ⁇ m filter membrane to obtain a pseudovirus solution, which was stored at -80°C.
  • TCID 50 values Use the limiting dilution method to perform 10-fold gradient dilution of the virus, set a total of 10 virus concentrations, each with 6 replicate wells.
  • the 96-well plate was seeded with a density of 3 ⁇ 10 4 cell/mL overexpressing ACE2 293FT cells (293FT-ACE2, source: Shenzhou Cell Engineering Co., Ltd., the same below) suspension, 100 ⁇ L/well.
  • Add 50 ⁇ L of the virus in gradient dilution to each well use the cell culture medium as a negative control, mix well, and place it in a 37°C, 5% CO 2 incubator for 24 hours. After incubation, add 5 ⁇ passive lysis buffer (source: Promega), 30 ⁇ L/well, and mix well to lyse the cells. Take 10 ⁇ L / white bottom 96 well bore into the fluorescent signal is detected, calculates Reed-Muench method TCID 50 values.
  • CoV2-mhB27 antibody 2000ng/mL, 571.4ng/mL, 163.3ng/mL, 46.6ng/mL, 13.3ng/mL, 3.8ng/mL, 1.1ng/mL, 0.31 ng/mL and 0.089ng/mL
  • 50 ⁇ L/well 50 ⁇ L/well.
  • 100 TCID 50 pseudovirus was used as a positive control, and the virus-free and antibody-free group was used as a negative control. After mixing, incubate in a 37°C, 5% CO 2 incubator for 1 hour.
  • the amino acid sequence of the heavy chain variable region (SEQ ID NO: 8) and the amino acid sequence of the light chain variable region (SEQ ID NO: 9) of the CoV2-mhB27 neutralizing antibody were deduced.
  • SEQ ID NO: 10-15 and Table 1 The light chain and heavy chain CDRs of the above antibodies are directly transplanted into the finally obtained humanized antibody CoV2-HB27 in the subsequent humanization step.
  • the classical CDR transplantation method is used to humanize the mouse antibody [8,9] .
  • the similarity with the murine light chain and heavy chain variable regions is more than 50%
  • the framework regions of the light chain and heavy chain variable regions are similar to the amino acid sequences of the framework regions of the light chain and heavy chain variable regions of the antibody to be modified
  • An antibody with a sex ratio of more than 50% is used as a humanized template, and the three CDR sequences of the light chain or heavy chain of the mouse antibody are replaced in the corresponding CDR amino acid sequence in the humanized template.
  • the human template for the light chain variable region transplantation of CoV2-mhB27 was selected as IGKV3D-11*02, which is similar to CoV2.
  • the homology of the light chain of mhB27 is 62.8%; the human template of the variable region of the heavy chain is IGHV3-21*01, which has a homology of 78.9% with the heavy chain of CoV2-mhB27.
  • the key points of the mouse-derived framework region play a vital role in maintaining the stability of the CDR spatial structure, the key points need to be backmutated to the corresponding amino acids of the mouse antibody.
  • the 42nd position of the light chain was backmutated to F
  • the 71th position was backmutated to V
  • the 83th position was backmutated to S
  • the 88th position was backmutated to S
  • the 103rd position was backmutated to F
  • the back mutation at position 3 was K
  • the back mutation at position 49 was R
  • the back mutation at position 54 was A
  • the back mutation at position 86 was T.
  • Humanized antibody CoV2-HB27 was obtained by CDR humanization transplantation and framework region back mutation.
  • the amino acid sequences of the heavy chain and light chain variable region are shown in SEQ ID NO: 22/23; its heavy chain containing signal peptide
  • the amino acid sequence of the light chain and the light chain are shown in SEQ ID NO: 18/19, respectively, which respectively include the heavy chain/light chain signal peptide amino acid sequence (SEQ ID NO: 20/21) connected in sequence; the humanized antibody heavy chain/light chain
  • the amino acid sequence of the variable region of the chain (SEQ ID NO: 22/23); the constant region of the humanized antibody is the human IgG1 heavy chain constant region/human kappa light chain constant region sequence (SEQ ID NO: 32/33).
  • the CoV2-HB27 heavy chain variable region nucleotide sequence (SEQ ID NO: 30) was obtained by the method of full gene synthesis. Inserted into the pSE vector digested with ScaI+NheI (source: Fermentas, the same below) with heavy chain signal peptide (SEQ ID NO: 28) and heavy chain IgG1 constant region (SEQ ID NO: 32) by In-fusion method The CoV2-HB27 heavy chain (SEQ ID NO: 26) expression vector was obtained.
  • the nucleotide sequence of CoV2-HB27 light chain variable region (SEQ ID NO: 31) was obtained by the method of whole gene synthesis. Inserted into pSE digested with ScaI+BsiWI (source: Fermentas) with light chain signal peptide (SEQ ID NO: 29) and light chain kappa constant region nucleotide sequence (SEQ ID NO: 33) by In-fusion method.
  • the CoV2-HB27 light chain (SEQ ID NO: 27) expression vector was obtained from the vector.
  • the plasmid was extracted and transfected into HEK-293 cells (source: Invitrogen, the same below), cultured and expressed for 7 days, and purified by a protein A purification column to obtain high-purity antibodies.
  • SARS-CoV-2 RBD protein 777.8ng/mL, 259.3ng/mL, 86.4ng/mL, 28.8ng/mL, 9.6ng/mL and 3.2ng/mL
  • S1 protein 21000ng/mL, 7000ng/mL, 2333.3ng/mL, 777.8ng/mL, 259.3ng/mL, 86.4ng/mL, 28.8ng/mL, 9.6ng/mL and 3.2ng/mL
  • ECD protein 21000ng/mL, 7000ng/mL , 2333.3 ng/mL, 777.8 ng/mL, 259.3 ng/mL, 86.4 ng/mL, 28.8 ng/mL and 9.6 ng/mL
  • Example 2.2 to detect the ability of the humanized antibody to compete for the binding of the ACE2 receptor to the SARS-CoV-2 RBD protein.
  • the biomolecule interaction analysis system (model: OctetRED96e, manufacturer: Fortebio) was used to determine the humanized antibody CoV2-HB27 and biotinylated SARS-CoV-2 RBD and S1 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) Binding affinity.
  • Select SA Sensor add 2 ⁇ g/mL of biotinylated SARS-CoV-2 RBD and S1 protein after 60s of equilibration, and then equilibrate again for 100s to wash away unbound protein.
  • CoV2-HB27 has high affinity with SARS-CoV-2 RBD and S1 protein.
  • the affinity of CoV2-HB27 to the SARS-CoV-2 RBD protein is 3.9E-11M, the binding constant is 3.7E+05 1/Ms, the dissociation constant is 1.4E-05 1/s, and R 2 is 1.000; and SARS- The affinity of CoV-2 S1 protein is 3.7E-10M, the binding constant is 1.1E+06 1/Ms, the dissociation constant is 3.9E-04 1/s, and R 2 is 0.997.
  • the specific kinetic characteristic parameter curve is shown in Figure 8.
  • CoV2-HB27 (500ng/mL, 200ng/mL, 80ng/mL, 32ng/mL, 12.8ng/mL, 5.12ng/mL, 2.05ng/mL, 0.82ng/mL and 0.33 ng/mL) the ability to neutralize 100TCID50SARS-CoV-2 pseudovirus in 293FT-ACE2 cells.
  • the results are shown in Figure 9.
  • CoV2-HB27 can effectively neutralize the pseudovirus of SARS-CoV-2 in a concentration-dependent manner, with a neutralization IC 50 of 5.9 ng/mL.
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • SEC-HPLC size-exclusion high performance liquid chromatograph
  • SEC-HPLC operation steps are: (1) Instrument: liquid chromatography system (Agilent company, model: Agilent1260), hydrophilic silica gel high-performance molecular exclusion chromatography column (Tosoh company, model: TSK-GEL G3000SW XL (7.8 ⁇ 300mm, 5 ⁇ m)); (2) Mobile phase: 200mM NaH 2 PO 4 , 100mM Arginine, pH 6.5; (2) Sample amount is 80 ⁇ g; (3) Detection wavelength is 280nM, analysis time is 30min, flow rate is 0.5mL/min , The column temperature is 25°C; (4) Calculate the ratio of each peak according to the area normalization method.
  • Instrument liquid chromatography system (Agilent company, model: Agilent1260), hydrophilic silica gel high-performance molecular exclusion chromatography column (Tosoh company, model: TSK-GEL G3000SW XL (7.8 ⁇ 300mm, 5 ⁇ m)); (2) Mobile phase: 200mM
  • the DLS test result of CoV2-HB27 showed that the radius was 5.8nM and the percent dispersion (%Pd) was 9.8%, indicating that the CoV2-HB27 particles were small, the size of normal IgG1 antibody particles, and the uniformity was good (Table 3).
  • DFS Differential scanning fluorimetry
  • Instrument Uncle system (Unchained Labs, Model: UNCLE-0330); (2) The sample volume is 9 ⁇ L; (3) The detection parameters are set: the temperature range is 25°C ⁇ 95°C, the heating speed Incubate at 0.3°C/min for 30s at each temperature; (4) Use UNcle Analysis software to analyze the data, take the midpoint value of the internal fluorescence curve under UV266 as T m , and take the aggregate formed by the static light scattering signal under UV266/Blue473 The polymerization start temperature of the change curve is Tagg 266 and Tagg 473.
  • IgG1 antibody has asparagine (Asn) deamidation, lysine (Lys) glycation, methionine (Met) oxidation, etc., so there will be uneven charge, showing acidic and basic isomers .
  • CEX-HPLC cation exchange high performance liquid chromatograph
  • cIEF capillary isoelectric focusing
  • CEX-HPLC operation steps (1) Instrument: liquid chromatography system (Agilent company, model: Agilent1260), cation exchange chromatography column (Thermo company, model: ProPac TM WCX-10 (4 ⁇ 250mm, 5 ⁇ M)); (2) ) Mobile phase A: buffer A, pH 5.6 (Thermo company, product number: 083273); (3) Mobile phase B: buffer B, pH 10.2 (Thermo company, product number: 083275); (4) The sample amount is 80 ⁇ g; ( 5) The detection wavelength is 280nM, the analysis time is 50min, the flow rate is 0.5mL/min, and the column temperature is 25°C; (6) The ratio of each peak is calculated by the area normalization method.
  • Instrument imaging capillary electrophoresis instrument (Proteinsimple, model: iCE3); (2) Take 10 ⁇ L of 5mg/ml sample and 8 ⁇ L Pharmalyte 3-10, 70 ⁇ L 1% methyl cellulose (MC), 2 ⁇ L PI marker And ddH 2 O was prepared into an analysis solution with a total volume of 200 ⁇ L; (3) The sample was placed in an iCE3 instrument, 1500V was pre-focused for 1 minute, and then 3000V was collected for 6 minutes; (4) Chrom Perfect software was used to analyze the data.
  • the CoV2-HB27 sample was stored at -80°C for 3 hours and then transferred to 45°C to thaw for 1 hour. The freeze-thaw cycle was repeated five times.
  • the 11.1mg/mL CoV2-HB27 sample was concentrated to 26.0mg/mL, 51.4mg/mL, 85.5mg/mL, 103.9mg/mL using a 50kDa ultrafiltration tube, and the purity of the sample was analyzed by SDS-PAGE and SEC-HPLC. Use DLS to analyze the change of sample particle size, and the specific steps are the same as 5.1.
  • Example 2.2 show that CoV2-mhB27 can compete for the binding of ACE2 receptor to SARS-CoV-2 RBD and S1 protein. Crystallization by cryo-electron microscopy showed that residues 437-508 are the key amino acid residues required for the binding of SARS-CoV-2 RBD to ACE2 [10] . Based on the above information, it is speculated that after CoV2-mhB27 is combined with RBD, it will form a steric conflict with the structural conformation of ACE2.
  • 17 residue sites located in the ACE2 binding region and its vicinity were selected and mutated into other residue types that are quite different from the original residue types, resulting in 15 mutants, namely E340R, T345Y, V367F, K378D, T385Y, N439R, N440Y, Y449A/N450, Y489R, T500Y, Y505E, A372Y, S375Y, D405R/R408, and V503Y.
  • SARS-CoV-2 RBD-His was used as a template (sequence source: https://www.gisaid.org/), PCR was used for site-directed mutation, and sequencing verification was performed.
  • the mutant and wild-type (WT) SARS-CoV-2 RBD proteins were transiently transfected and the binding ability of CoV2-mhB27 antibody to the mutant protein was detected by ELISA.
  • the ELISA readings of WT SARS-CoV-2 RBD were used as the standard to standardize the test results of each antibody.
  • the highest antibody binding reading (set as 100%) was used to further normalize the ELISA binding signal for the mutant.
  • the ELISA binding signal of mhB27 for a specific mutant drops below 75% relative to WT SARS-CoV-2 RBD, this residue site is defined as a significant binding epitope.
  • the ELISA binding signal of CoV2-mhB27 against a specific mutant drops below 50%, it is defined as a highly significant epitope.
  • N439 and T500 in SARS-CoV-2 RBD are highly significant epitopes of CoV2-mhB27.
  • nucleotide mutations in the constant region of the IgG1 subtype were carried out with reference to the literature [11] to obtain the genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fd6-IgG1, SEQ ID NO:44).
  • the CoV2-HB27-Fd6-IgG1 heavy chain sequence (SEQ ID NO: 46) was obtained by PCR, which includes the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 28), and the heavy chain variable region nucleotide sequence (SEQ ID NO: 28). ID NO: 30) and Fd6-IgG1 constant region nucleotide sequence (SEQ ID NO: 44).
  • the expression vector containing CoV2-HB27-Fd6-IgG1 heavy chain (SEQ ID NO: 46) was inserted into the pSE vector digested with HindIII+XbaI by In-fusion method.
  • CoV2-HB27-Fd6-IgG1 heavy chain (SEQ ID NO: 46) expression vector and CoV2-HB27 light chain (SEQ ID NO: 27) expression vector plasmids were extracted, transfected into HEK-293 cells and cultured and expressed for 7 days, using protein Purification by purification column A obtains CoV2-HB27-Fd6-IgG1 antibody that reduces Fc function.
  • CoV2-HB27 heavy chain (SEQ ID NO: 26) expression vector and CoV2-HB27 light chain (SEQ ID NO: 27) expression vector plasmids were extracted, transfected into HEK-293 (fut8 gene knockout) cells and cultured and expressed for 7 days. Purified by a protein A purification column, the CoV2-HB27-Ae0-IgG1 antibody of defucosylated IgG1 subtype was obtained.
  • nucleotide mutations in the constant region of the IgG1 subtype were carried out with reference to the literature [12,13] to obtain the genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fe4-IgG1, SEQ ID NO: 48).
  • the CoV2-HB27-Fe4-IgG1 heavy chain sequence (SEQ ID NO: 50) was obtained by PCR, which contains the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 28), and the heavy chain variable region nucleotide sequence (SEQ ID NO: 28). ID NO: 30) and Fe4-IgG1 constant region nucleotide sequence (SEQ ID NO: 48).
  • the CoV2-HB27-Fe4-IgG1 heavy chain (SEQ ID NO: 50) expression vector was inserted into the pSE vector digested with HindIII+XbaI by In-fusion method.
  • CoV2-HB27-Fe4-IgG1 heavy chain (SEQ ID NO: 50) expression vector and CoV2-HB27 light chain (SEQ ID NO: 27) expression vector plasmids were extracted and transfected into HEK-293 (fut8 gene knockout) cells for culture After 7 days of expression, the protein A purification column was used to obtain CoV2-HB27-Fe4-IgG1 antibody with enhanced Fc function.
  • Example 8 Fc function of different forms of humanized antibody CoV2-HB27
  • Avidin protein (source: Thermo, the same below) at a concentration of 10 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 2-8°C. Wash the plate the next day. After blocking at room temperature for 1 hour, add 100 ⁇ L of 5 ⁇ g/mL biotin-labeled CD16a-AVI-His(V158)+BirA protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), incubate at room temperature for 1 hour and then wash the plate .
  • Avidin protein at a concentration of 10 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 2-8°C. Wash the plate the next day and block for 1 hour at room temperature.
  • CoV2-HB27 antibodies with different Fc functional forms were added, and the antibody concentrations were 40000ng/mL, 10000ng/mL, 2500ng/mL, 625ng/mL, 156.3ng/mL, 39.1ng/mL and 9.77ng/mL.
  • the plate was washed to remove unbound antibodies, and goat anti-human IgG F(ab)2/HRP was added to incubate and the plate was washed repeatedly, and the substrate color development solution was added for color development. After termination, the microplate reader reads OD 450 .
  • Avidin protein at a concentration of 10 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 2-8°C. The plate was washed the next day and sealed at room temperature for 1 hour, and 100 ⁇ L of biotin-labeled CD64-AVI-His+BirA protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added at a concentration of 0.5 ⁇ g/mL, and the plate was washed after incubating for 1 hour at room temperature.
  • CoV2-HB27 antibodies with different Fc functional forms Add 100 ⁇ L of CoV2-HB27 antibodies with different Fc functional forms, and the antibody concentration is 2500ng/mL, 625ng/mL, 156.3ng/mL, 39.1ng/mL, 9.77ng/mL, 2.44ng/mL, 0.61ng/mL, 0.15 ng/mL, 0.04ng/mL and 0.01ng/mL.
  • the plate was washed to remove unbound antibodies, and goat anti-human IgG F(ab)2/HRP was added to incubate and the plate was washed repeatedly. Finally, the substrate color solution was added for color development. After termination, the plate reader reads OD 450 .
  • the Fe4-IgG1 form antibody with enhanced Fc function has the strongest binding ability to CD64, which is better than the defucosylated Ae0-IgG1 form antibody and CoV2-HB27 antibody of IgG1 subtype; Fd6-IgG1 antibody with reduced Fc function only weakly binds to CD64.
  • CoV2-HB27 antibodies with different concentrations of different Fc functional forms were respectively coated on 96-well plates, 100 ⁇ L/well, coated overnight at 4°C, antibody coating concentrations were 40000ng/mL, 10000ng/mL, 2500ng/mL, 625ng /mL, 156.3ng/mL, 39.1ng/mL and 9.77ng/mL.
  • the plate was washed the next day and sealed at room temperature for 1 hour, then 5 ⁇ g/mL of C1q complement protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100 ⁇ g/well, and incubated for 1 hour.
  • the Fe4-IgG1 form antibody with enhanced Fc function has the strongest binding ability to C1q, which is better than the defucosylated Ae0-IgG1 form antibody and CoV2-HB27 antibody of IgG1 subtype; Fd6-IgG1 antibody with reduced Fc function only weakly binds to C1q.
  • the HEK293FT monoclonal cell line stably expressing the full-length SARS-CoV-2 protein (HEK293FT-SARS-CoV-2-S, source: Shenzhou Cell Engineering Co., Ltd., the same below) was used as the target cell to stably transfect CD16AV and NFAT -Luc2P Jurkat cells (Jurkat-NFAT/Luc2P-CD16AV) are effector cells, and the ADCC function of humanized antibodies is detected by the reporter gene method.
  • target cells with a density of 1 ⁇ 10 5 cell/mL and effector cells with an equal volume and an equal density were inserted at 50 ⁇ L/well. Then add 50 ⁇ L of CoV2-HB27 antibody and H7N9-R1 negative control antibody in different Fc functional forms.
  • CoV2-HB27-Ae0-IgG1 CoV2-HB27-He5-IgG1 antibody was added at a concentration of 6.25ng/mL, 0.78ng/mL, 0.097ng/mL, 0.012ng/mL, 0.0015ng/mL and 0.00019ng/mL, the rest The antibody concentration was 50ng/mL, 6.25ng/mL, 0.78ng/mL, 0.097ng/mL, 0.012ng/mL, 0.0015ng/mL and 0.00019ng/mL. After mixing, incubate in a 37°C, 5% CO 2 incubator for 6 hours. Finally, add 5 ⁇ passive lysis buffer, 30 ⁇ L/well, and mix well to lyse the cells.
  • the Fe4-IgG1 form antibody with enhanced Fc function can mediate the strongest ADCC effect, which is better than the defucosylated Ae0-IgG1 form antibody And IgG1 subtype antibody mediated ADCC effect; Fd6-IgG1 form antibody that reduces Fc function has no ADCC effect.
  • Jurkat cells With HEK293FT-SARS-CoV-2-S as target cells, Jurkat cells (Jurkat-NFAT/Luc2P-CD32A, Jurkat-NFAT/Luc2P-CD32B or Jurkat-NFAT) stably transfected with CD32A, CD32B or CD64 and NFAT-Luc2P /Luc2P-CD64) are effector cells, and the reporter gene method is used to detect the ADCP function mediated by humanized antibodies.
  • target cells with a density of 1 ⁇ 10 5 cell/mL and effector cells with an equal volume and an equal density were inserted at 50 ⁇ L/well. Then add 50 ⁇ L of CoV2-HB27 antibody and H7N9-R1 negative control antibody in different Fc functional forms.
  • the antibody concentration is 50ng/mL, 6.25ng/mL, 0.78ng/mL, 0.097ng/mL, 0.012ng/mL, 0.0015ng /mL, 0.00019ng/mL, 0.000024ng/mL and 0.000003ng/mL;
  • the antibody concentration is 6.25ng/mL, 0.78ng/mL, 0.097ng/mL, 0.012 ng/mL, 0.0015ng/mL, 0.00019ng/mL, 0.0000024ng/mL and 0.000003ng/mL.
  • WST-8 method was used to detect the CDC function of humanized antibodies.
  • target cells with a density of 2 ⁇ 10 6 cells/mL were inserted at 50 ⁇ L/well.
  • 50 ⁇ L of rabbit complement (source: One lambda) and CoV2-HB27 antibodies of different Fc functional forms and set up detection blank holes (no cells), positive control (only inoculated cells) control and H7N9-R1 negative control antibody groups antibodies
  • the added concentrations were 20ng/mL, 4ng/mL, 0.8ng/mL, 0.16ng/mL, 0.032ng/mL, 0.0064ng/mL and 0.00128ng/mL.
  • Example 9 Evaluation of mouse pharmacokinetics of humanized antibody CoV2-HB27 with different Fc functional forms
  • mice choose C57BL/6 mice (a total of 6 mice, male and female, source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), a single tail vein injection of CoV2-HB27, the dose of 5mg/kg, the volume of administration It is 10mL/kg.
  • 30min, 1h, 3h, 6h, 10h, 24h, 32h, 48h, 72h, 96h, 120h, 168h, 240h, 336h, 504h and 672h after administration all mice were subjected to orbital blood sampling and centrifugation Take the serum.
  • the blood drug concentration was detected by ELISA method, and the non-compartmental model (NCA) in Phoenix-WinNonlin 8.1 software was used to calculate the pharmacokinetic parameters.
  • NCA non-compartmental model
  • C57BL/6 mice (a total of 6 mice, half male and half male, source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected to give CoV2-HB27-Fd6-IgG1 antibody by a single tail vein injection at a dose of 5 mg/ kg, the administration volume is 10mL/kg.
  • 30min, 1h, 3h, 6h, 10h, 24h, 32h, 48h, 72h, 96h, 120h168h, 240h, 336h, 504h and 672h after administration blood was collected from the orbit of all mice, and serum was collected by centrifugation .
  • the blood drug concentration was detected by ELISA, and the non-compartmental model (NCA) in Phoenix-WinNonlin 6.4 software was used to calculate the pharmacokinetic parameters.
  • NCA non-compartmental model
  • mice were in normal condition, and the drug-time curve is shown in Figure 19.
  • the drug concentration in the mice continued to decrease over time, with a rapid decrease in the early stage, but a slower change in a longer period of time, and there was no obvious gender difference.
  • the pharmacokinetic parameters are shown in Table 10.
  • the average in vivo exposure C max and AUC last were 115.04 ⁇ g/mL and 11159.82h ⁇ g, respectively /mL, the average half-life t 1/2 is 320.00h, and the clearance rate Cl is 0.23mL/h/kg.

Abstract

本发明属于细胞免疫技术领域，提供了一种SARS-CoV-2中和人源化单克隆抗体。该抗体可封闭SARS-CoV-2刺突蛋白（S蛋白）同ACE2受体的结合，高效中和SARS-CoV-2病毒侵染细胞；制备的人源化中和抗体可作为特异性抗体药物用于预防和治疗SARS-CoV-2病毒引起的急性呼吸道传染病。

Description

[根据细则37.2由ISA制定的发明名称]　SARS-COV-2中和抗体及其制备和应用
相关申请的交叉引用
本申请要求2020年04月28日提交的中国专利申请202010349190.3的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及细胞免疫技术领域，提供可封闭SARS-CoV-2刺突蛋白(S蛋白)同ACE2受体的结合、高效中和SARS-CoV-2病毒侵染细胞的人源化抗体。其可用于治疗SARS-CoV-2引起的感染性疾病。本发明还提供了编码所述抗体的核酸序列、含有所述核酸序列的载体、细胞。
背景技术
SARS-CoV-2在人与人之间的传播途径与SARS-CoV(Severe Acute Respiratory Syndrome coronavirus)及MERS-CoV(Middle East Respiratory Syndrome)相似，主要为呼吸道飞沫传播，亦可通过接触传播。SARS-CoV-2主要传染源为COVID-19患者，无症状感染者也可能成为传染源。相关研究表明SARS-CoV-2的基本传染数(R0值)在2.24至3.58之间，提示其具有较强的传播能力 [1]。人群对SARS-CoV-2普遍易感，潜伏期为1-14天，多为3-7天。
新型冠状病毒肺炎诊疗方案(试行第七版)》将COVID-19分为轻型、普通型、重型和危重型。COVID-19的临床症状以发热、干咳、乏力为主，少数患者伴有鼻塞、流涕、咽痛、肌痛和腹泻等症状。轻型患者仅表现为低热，轻微乏力等，无肺炎表现。重症患者多表现为在发病一周后出现呼吸困难和/或低氧血症，严重者可快速进展为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍及多器官功能衰竭等 [2,3]。
由于暴发突然、疫情传播迅速、应对时间有限，目前尚无针对SARS-CoV-2感染特异性的有效临床治疗方案，临床上仅能对症支持治疗，多以缓解症状、预防继发感染、减少并发症发生以及进行器官功能支持为主。许多治疗药物选择都来自SARS-CoV、MERS-CoV等感染的临床治疗经验，也有一些候选药物如瑞德西韦(一种核苷酸类似物，可抑制RNA聚合酶，阻断病毒复制)、洛匹那韦/利托那韦(克力芝)、干扰素、磷酸氯喹等正在进行临床试验。
SARS-CoV-2与SARS-CoV具有共同的宿主细胞受体蛋白，即血管紧张素转化酶2(ACE2) [4]。病毒的三聚体刺突蛋白(S蛋白)同ACE2受体结合后被宿主蛋白酶切割为包含受体结合域(Receptor binding domain,RBD)的S1多肽和负责介导病毒同细胞膜融合的S2多肽，进而侵入体内 [5]。因而，寻找和制备有效的抗体阻止SARS-CoV-2 RBD蛋白和ACE2受体结合，进而抑制病毒侵染细胞，成为预防和治疗SARS-CoV-2病毒感染的重中之重。大量体外研究表明，针对SARS-CoV、MERS-CoV和Ebola等病毒表面蛋白受体结合区域的中和抗体，可有效阻断病毒与受体的结合，从而阻断病毒入侵宿主细胞。目前，临床上已经使用病人恢复期血浆治疗SARS-CoV-2的探索，并取得积极的治疗效果。但是，恢复期血浆来源有限，操作复杂，成本大且存在安全性等问题，如增加抗体依赖的增强作用(Antibody dependent enhancement，ADE)等风险。通过制备抗SARS-CoV-2 RBD蛋白单克隆抗体，筛选可与之特异性结合的中和抗体，进一步对其人源化已成为制备预防或治疗性抗体药物的有效手段。
由于尚无针对SARS-CoV-2感染的治疗药物，目前对于COVID-19的治疗方案多以缓解症状、预防继发感染、减少并发症发生以及进行器官功能支持为主。因此本领域迫切需要开发具有良好中和病毒效果的高亲和力的冠状病毒中和性抗体，尤其是单克隆抗体。本 专利发明的人源化单克隆抗体能高效中和SARS-CoV-2病毒，可作为特异性抗体药物用于预防和治疗SARS-CoV-2病毒引起的急性呼吸道传染病。
发明内容
在一个方面，本发明提供一种分离的、封闭SARS-CoV-2刺突蛋白同ACE2受体的结合抗体或其抗原结合片段，其包含
i)重链可变区，其重链CDR1、重链CDR2和重链CDR3域分别包含SEQ ID NO:13、14和15，和/或
ii)轻链可变区，其轻链CDR1、轻链CDR2和轻链CDR3域分别包含SEQ ID NO:10、11和12
在一个实施方式中，所述抗体或其抗原结合片段包含：
i)重链可变区，其序列包含SEQ ID NO:22或与其具有至少85％、88％、90％、95％、98％或99％序列同一性；和/或
ii)轻链可变区，其序列包含SEQ ID NO:23或与其具有至少85％、88％、90％、95％、98％或99％序列同一性。
在一个实施方式中，所述抗体或其抗原结合片段为人源化抗体、嵌合或鼠源抗体。
在一个实施方式中，所述抗体或其抗原结合片段，其抗体恒定区为IgG，IgM，IgA亚型，优选地，为IgG1、IgG2或IgG4亚型抗体；更优选地，为因其Fc区的氨基酸序列和/或糖基化形式改变造成其与Fc受体和/或C1q补体结合功能改变的IgG1、IgG2或IgG4亚型抗体。
在一个实施方式中，所述抗体或其抗原结合片段，
a)其与SARS-CoV-2 RBD的结合亲和力K D平均值为11.7E-11～1.3E-11M，优选5.9E-11～2.6E-11M,更优选为3.9E-11M；和/或
b)其与SARS-CoV S1的结合亲和力K D平均值为11.1E-10～1.2E-10M，优选5.6E-10～2.5E-10M,更优选为3.7E-10M。
在一个实施方式中，所述抗体或其抗原结合片段表位为SARS-CoV-2病毒刺突蛋白的N439和T500。
在一个实施方式中，所述抗体或其抗原结合片段进一步包含:
i)重链恒定区，优选地，其序列包含SEQ ID NO:24或与其具有至少90％、92％、95％、98％或99％序列同一性；和/或
ii)轻链恒定区,优选地，其序列包含SEQ ID NO:25或与其具有至少90％、92％、95％、98％或99％序列同一性。
在一个实施方式中，所述抗体经单次静脉注射给予小鼠后，在体内的平均暴露量C max和AUC last分别为108.96μg/mL和9922.11h×μg/mL，平均半衰期t 1/2为286.16h，清除率Cl为0.30mL/h/kg。
在一个实施方式中，所述抗体由fut8基因敲除的HEK-293表达。
在一个实施方式中，所述抗体进一步包含:
i)重链恒定区，优选地，其序列包含SEQ ID NO:45或与其具有至少90％、92％、95％、98％或99％序列同一性；和/或
ii)轻链恒定区,优选地，其序列包含SEQ ID NO:25或与其具有至少90％、92％、95％、98％或99％序列同一性。
在一个实施方式中，所述的抗体，其
同CD16a仅有极弱的结合；
同CD32a和CD32b几乎无结合；
同CD64仅有较弱的结合；
无ADCC作用；
无ADCP作用；
无CDC作用。
在一个实施方式中，所述抗体经单次静脉注射给予小鼠后，在体内的平均暴露量C max和AUC last分别为115.04μg/mL和11159.82h×μg/mL，平均半衰期t 1/2为320.00h，清除率Cl为0.23mL/h/kg。
在一个实施方式中，所述抗体由fut8基因敲除的HEK-293表达。
在一个实施方式中，所述的抗体，其
同CD16a的结合能力强于IgG1亚型的CoV2-HB27抗体；
显著介导对表达SARS-CoV-2S的ADCC作用。
在一个实施方式中，所述抗体进一步包含
i)重链恒定区，优选地，其序列包含SEQ ID NO:49或与其具有至少90％、92％、95％、98％或99％序列同一性；和/或
ii)轻链恒定区,优选地，其序列包含SEQ ID NO:25或与其具有至少90％、92％、95％、98％或99％序列同一性。
在一个实施方式中，所述抗体由fut8基因敲除的HEK-293表达。
在一个实施方式中，所述的抗体，其
同CD16a的结合能力强于IgG1亚型的CoV2-HB27抗体；
同CD32a和CD32b强于IgG1亚型的CoV2-HB27抗体；
同CD64的结合能力强于IgG1亚型的CoV2-HB27抗体；
同C1q的结合能力强于IgG1亚型的CoV2-HB27抗体；
介导ADCC作用强于IgG1亚型的CoV2-HB27抗体；
在高浓度下可以介导较弱的ADCP作用；
在一个实施方式中，所述的抗体或其抗原结合片段为单克隆抗体。
在一个实施方式中，所述抗原结合片段为Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子或单域抗体；其中单链抗体分子优选为scFv、di-scFv、tri-scFv、双体抗体或scFab。
在另一个方面，本发明提供一种改构抗体-药物分子，其包含本发明的抗体或其抗原结合片段和与之共价或非共价连接的小分子或生物大分子，优选地通过接头连接。
在又一个方面，本发明提供一种核酸，其编码根据本发明所述的抗体或其抗原结合片段，其为mRNA和/或DNA。
在一个实施方式中，所述的核酸，其包含
i)分别如SEQ ID NO:30所示的重链可变区核苷酸序列和/或如SEQ ID NO:31所示的轻链可变区核苷酸序列；和任选地
ii)分别如SEQ ID NO:32、44、48所示重链恒定区核苷酸序列和/或如SEQ ID NO:7所示的轻链恒定区核苷酸序列；
或i)和ii)的变体。
在又一个方面，本发明提供一种表达载体，其包含如本发明所述的核酸。
在又一个方面，本发明提供一种宿主细胞，其包含如本发明所述的核酸或如本发明所述的表达载体。
在又一个方面，本发明提供一种用于产生如本发明所述的抗体或其抗原结合片段的方法，其包括在适合于抗体表达的条件下培养如本发明所述的宿主细胞，和从培养基中回收表达的抗体。
在又一个方面，本发明提供一种药物组合物，其包含
如本发明所述的抗体或其抗原结合片段或如本发明所述的改构抗体-药物分子或如本发明所述的核酸或如本发明所述的表达载体；
药学上可接受的载体；任选地
一种或多种其他治疗剂，优选地，其他治疗剂选自抗病毒药物或炎性因子抑制剂、其他机制的小分子化学药；优选地，抗病毒药物选自包含不限于I型干扰素药物、抗体类、蛋白酶抑制剂类、RNA依赖性RNA聚合酶(RdRP)抑制剂类、靶向宿主的抗病毒类药物。
在又一个方面，如本发明所述的抗体或其抗原结合片段、如本发明所述的改构抗体-药物分子、如本发明所述的核酸、如本发明所述的表达载体、如本发明所述的药物组合物，其用于预防和治疗SARS-CoV-2感染引起的疾病。
在又一个方面，如本发明所述的抗体或其抗原结合片段、如本发明所述的改构抗体-药物分子、如本发明所述的核酸、如本发明所述的表达载体、如本发明所述的药物组合物在用于制备用于预防和治疗SARS-CoV-2感染引起的疾病的药物中的应用。
在又一个方面，本发明提供一种药物组合，其包含
如本发明所述的抗体或其抗原结合片段、如本发明所述的改构抗体-药物分子、如本发明所述的核酸、如本发明所述的表达载体、如本发明所述的药物组合物；以及一种或多种另外的治疗剂。
在又一个方面，本发明提供一种试剂盒，其包含
如本发明所述的抗体或其抗原结合片段、如本发明所述的改构抗体-药物分子、如本发明所述的核酸、如本发明所述的表达载体、如本发明所述的药物组合物；优选地，还进一步包含给药的装置。
在又一个方面，本发明提供一种预防和治疗SARS-CoV-2感染引起的疾病的方法，其包含给予受治疗者如本发明所述的抗体或其抗原结合片段、如本发明所述的改构抗体-药物分子、如本发明所述的核酸、如本发明所述的表达载体、如本发明所述的药物组合物、如本发明所述的药物组合、或如本发明所述的试剂盒。
在又一方面，本发明提供一种分离的、封闭SARS-CoV-2刺突蛋白同ACE2受体的结合抗体或其抗原结合片段，其结合表位是SARS-CoV-2刺突蛋白的N439和T500。
在又一方面，本发明提供一种SARS-CoV-2刺突蛋白的结合表位，其为SARS-CoV-2刺突蛋白的N439和T500。
附图说明
图1：结合SARS-CoV-2蛋白单克隆噬菌体的筛选。
图2：鼠源抗体同SARS-CoV-2 S1及ECD蛋白的结合能力。
图3：流式检测鼠源抗体同SARS-CoV-2 S1蛋白的结合。
图4：鼠源抗体竞争ACE2与SARS-CoV-2 RBD和S1蛋白的结合。
图5：鼠源抗体中和SARS-CoV-2假病毒。
图6：人源化抗体同SARS-CoV-2 RBD，S1及ECD蛋白的结合能力。
图7：人源化抗体竞争ACE2蛋白与SARS-CoV-2 S1蛋白的结合。
图8：人源化抗体同SARS-CoV-2 RBD蛋白及S1蛋白的亲和力检测。
图9：人源化源抗体中和SARS-CoV-2假病毒。
图10：CoV2-mhB27表位分析的表位示意图(A)和ELISA检测结果(B)。A中SARS-CoV-2RBD以白色表面模型表示，所有设计的突变位点以浅灰色表示，鉴定所得的mhB27高度显著表位以黑色表示。
图11：不同Fc功能形式CoV2-HB27抗体同CD16a的结合。
图12：不同Fc功能形式CoV2-HB27抗体同CD32的结合。
图13：不同Fc功能形式CoV2-HB27抗体同CD64的结合。
图14：不同Fc功能形式CoV2-HB27抗体同C1q的结合。
图15：不同Fc功能形式CoV2-HB27抗体对表达SARS-CoV-2 S蛋白靶细胞介导的ADCC作用。
图16：不同Fc功能形式CoV2-HB27抗体对表达SARS-CoV-2 S蛋白靶细胞介导的ADCP作用。
图17：不同Fc功能形式CoV2-HB27抗体对表达SARS-CoV-2 S蛋白靶细胞介导的CDC作用。
图18：小鼠单次静脉注射CoV2-HB27后血药浓度均值-时间曲线(n＝6)。
图19：小鼠单次静脉注射CoV2-HB27-Fd6-IgG1后血药浓度均值-时间曲线(n＝6)。
具体实施方式
定义
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，进一步定义以下术语。
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
术语“抗体”意指免疫球蛋白分子，是指表现所需生物学活性的抗体的任何形式。包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体),甚至包括抗体片段。典型地，全长抗体结构优选包含4条多肽链，通常通过二硫键相互连接的2条重(H)链和2条轻(L)链。每条重链包含重链可变区和重链恒定区。每条轻链包含轻链可变区和轻链恒定区。在此典型全长抗体结构外,其结构还包括其他衍生形式。
术语“可变区”指抗体重链或轻链中涉及抗体结合抗原的域。天然抗体的重链和轻链的可变区(分别为VH和VL)一般具有类似的结构,可进一步细分为穿插在更保守的区域(称为框架区(FR))中的高变区(称为互补决定区(CDR))。
术语“互补决定区”(CDR，例如CDR1、CDR2和CDR3)是指抗体可变区的这样一些氨基酸残基，其存在对于抗原结合来说是必需的。每个可变区通常具有3个被鉴别为CDR1、CDR2和CDR3的CDR区域。每个互补决定区可包含来自如Kabat所定义的“互补决定区”的氨基酸残基(Kabat et al.,Sequences of Proteins of Immulological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD.1991))和/或来自“高变环”的那些残基(Chothia and Lesk；J Mol Biol 196:901-917(1987))。
术语“构架”或“FR”残基是如本文中所定义的CDR残基之外的那些可变区残基。
每个重链可变区和轻链可变区通常包含3个CDR和最多达4个FR，所述CDR和FR从氨基末端至羧基末端以例如以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
给定抗体的互补性决定区(CDR)和框架区(FR)可以使用Kabat体系标识(Kabat等:Sequences of Proteins of Immunological Interest，第5版，美国卫生和公众服务部，PHS，NIH，NIH出版编号91-3242，1991)。
术语“恒定区”是指抗体的轻链和重链上的这样一些氨基酸序列，不直接参与抗体与抗原的结合，但展现出多种效应子功能，例如抗体依赖性细胞毒性。
根据其重链恒定区的氨基酸序列，完整的抗体可归属于IgA、IgD、IgE、IgG和IgM五类抗体，其中IgG和IgA还可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。相应地，五类抗体的重链分别归入α、δ、ε、γ和μ链。根据其轻链恒定区的氨基酸序列，抗体的轻链可归入κ和λ。。
“抗体的抗原结合片段”包含完整抗体分子的一部分,其保留母体抗体的至少某些结合特异性，通常包括至少部分母体抗体的抗原结合区或可变区(例如一个或多个CDR)。抗原结合片段的实例包括但不限于Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子(例如scFv，di-scFv或tri-scFv、双体抗体或scFab)、单域抗体。
“抗体片段”是保留母体抗体的至少某些生物学特性的非完整抗体分子，其实例除上述 “抗原结合片段”所述及的那些之外,还包括但不限于Fc片段。
术语“改构药物分子”是指抗体或其片段,如抗原结合片段与另一分子形成共价或非共价连接物或形成重组多靶点融合药物,另一分子选自小分子化合物或生物大分子。
术语“嵌合”抗体是指重链和/或轻链的一部分来源于特定来源或物种，而其余部分来源于不同来源或物种的抗体。“人源化抗体”是“嵌合抗体”的子集。
术语“人源化抗体”或“人源化抗原结合片段”在本文中被定义为这样的抗体或抗体片段：(i)来源于非人来源(例如，携带异源免疫系统的转基因小鼠)且基于人种系序列的抗体；或(ii)可变区是非人来源而恒定区是人来源的嵌合抗体；或者(iii)CDR移植的，其中可变区的CDR来自非人来源，而可变区的一个或多个构架区为人来源的，并且恒定区(如果有的话)是人来源的。“人源化”的目的是消除非人来源抗体在人体内的免疫原性,而同时最大可能地保留亲和力。选择与非人来源抗体构架序列最相似的人构架序列为模板进行人源化改造是有利的。在某些情况下，可能需要用非人构架中相应的残基替换人类构架序列中的一个或多个氨基酸，以避免亲和性的丧失。
“单克隆抗体”是指获自基本上同质的抗体群体的抗体，即，所述包含单一抗体的群体除了可能以极少量存在的可能突变(例如天然突变)之外是相同的。因此，所述术语“单克隆”表明所述抗体的性质，即不是不相关抗体的混合物。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂的每个单克隆抗体均针对抗原上的单独一组决定簇。除了其特异性之外，单克隆抗体制剂的优点在于它们通常不会被其他抗体污染。所述术语“单克隆”不应被理解为需要通过任何特定的方法产生所述抗体。所述术语单克隆抗体具体地包括嵌合抗体、人源化抗体和人抗体。
抗体“特异性结合”目的抗原例如病毒相关抗原蛋白(本文中，刺突蛋白S)，即以足够的亲和力结合所述抗原以使得所述抗体可用作治疗剂，靶向表达所述抗原的病毒或细胞，并且与其他蛋白质无显著交叉反应或者与除了上文提到的抗原靶的同源体和变体(例如突变形式、剪接变体，或蛋白水解作用截短的形式)以外的蛋白质无显著交叉反应。
术语“结合亲和力”是指分子的单个结合位点与其结合伴侣之间非共价相互作用总和的强度。除非另有说明，用于本文时“结合亲和力”是指固有的结合亲和力，其反映结合对(例如抗体和抗原)的成员之间1:1的相互作用。“KD”、“结合速率常数kon”和“解离速率常数koff”通常用于描述分子(例如抗体)与其结合伴侣(例如抗原)之间的亲和力，即，配体结合特定蛋白的紧密程度。结合亲和力受非共价分子间相互作用的影响，例如氢键，静电相互作用，两个分子之间的疏水和范德华力。另外，配体与其靶分子之间的结合亲和力可能受到其他分子的存在的影响。亲和力可通过本领域中已知的常规方法来分析，包括本文描述的ELISA。
术语“表位”包括能够特异性结合至抗体或T细胞受体的任何蛋白质决定簇。表位决定簇通常由分子的化学活性表面基团(例如氨基酸或糖侧链，或其组合)组成，并且通常具有特定三维结构特征以及特定的电荷特征。
“分离的”抗体是已经被鉴别并且从天然表达该抗体的细胞中分离的抗体。分离的抗体包括重组细胞内的原位抗体以及通常通过至少一个纯化步骤进行制备的抗体。
两条多肽或核酸序列之间的“序列同一性”表示所述序列之间相同的残基的数目占残基总数的百分比。在计算同一性百分数时，将正在比较的序列以产生序列之间最大匹配的方式比对，通过特定算法解决比对中的空位(如果存在的话)。确定两个序列之间同一性的优选计算机程序方法包括，但不限于，GCG程序包，包括GAP、BLASTP、BLASTN和FASTA(Altschul等人，1990，J.Mol.Biol.215：403-410)。上述程序可以公开地从国际生物技术信息中心(NCBI)和其他来源得到。熟知的Smith Waterman算法也可用于确定同一性。
术语“受体”，是一个生物化学上的概念，指一类能传导细胞外信号，并在细胞内产生特定效应的分子。产生的效应可能仅在短时间内持续，比如改变细胞的代谢或者细胞的运动。也可能是长效的效应，比如上调或下调某个或某些基因的表达。
术语“Fc受体”或“FcR”指与抗体Fc区结合的受体。优选天然序列的人FcR，且优选与IgG抗体结合的受体(γ受体)，其包括FcγRI，FcγRII和FcγRIII亚型，以及这些受体的变体。其它FcR均被包含在术语“FcR”中。该术语也包括新生儿受体(FcRn)，其负责将母体的IgG转运至胎儿(Guyer等，免疫学杂志117：587(1976)和Kim等，免疫学杂志24：249(1994))。
术语“新生儿Fc受体”、简称“FcRn”，其结合IgG抗体Fc区。新生儿Fc受体(FcRn)在体内IgG类抗体的代谢命运中起重要作用。FcRn行使功能以从溶酶体降解途径营救IgG，从而降低其在血清中的清除率并加长半衰期。因此，IgG体外FcRn结合性质/特征指示它在血液循环中的体内药代动力学性质。
术语“效应子功能”指可归因于抗体的Fc区的那些生物学活性，其随抗体同种型而不同。抗体效应子功能的实例包括：C1q结合和依赖补体的细胞毒性(CDC)、Fc受体结合、“抗体依赖性细胞介导的细胞毒性”(ADCC)、抗体依赖性细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞对抗原的摄取、细胞表面受体(例如B细胞受体)的下调和B细胞激活。
术语“效应细胞”指表达一种或多种FcR并行使效应子功能的白细胞。在一个方面，所述效应细胞至少表达FcγRIII并执行ADCC效应子功能。介导ADCC的人白细胞的实例包括外周血单核细胞(PBMC)、自然杀伤(NK)细胞、单核细胞、细胞毒性T细胞和嗜中性粒细胞。效应细胞可以从天然来源，例如，血液中分离。效应细胞通常是与效应子阶段相关联的淋巴细胞，并发挥作用，以产生细胞因子(辅助T细胞)、杀死被病原体感染的细胞(细胞毒性T细胞)或分泌抗体(分化的B细胞)。
“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞。免疫细胞包括：淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
“抗体依赖性细胞介导的细胞毒性”或“ADCC”是指一种细胞毒性形式，其中结合到在某些细胞毒性细胞(例如NK细胞、嗜中性粒细胞和巨噬细胞)上存在的Fcγ受体上的分泌Ig使得这些细胞毒性效应细胞能够特异性结合至承载抗原的靶细胞，随后使用例如细胞毒素杀死所述靶细胞。为了评估目的抗体的ADCC活性，可进行体外ADCC测定法，例如记载于美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中的体外ADCC测定法、本申请的实施例中记载的方法。用于这类测定法的有用效应细胞包括PBMC和NK细胞。
“补体依赖性细胞毒性”或“CDC”是指在补体的存在下靶细胞的裂解。经典补体途径的活化由补体系统的第一组分(C1q)与(适当亚类的)抗体结合起始，其中该抗体与其相应抗原结合。为了评估补体活化，可进行CDC测定法，例如记载于Gazzano-Santoro et al.,J.Immunol Methods 202:163(1996)中的CDC测定法、例如本申请的实施例中记载的方法、例如在美国专利No.6,194,551Bl和WO1999/51642中记载的方法，其中描述了具有改变的Fc区氨基酸序列的多肽变体(具有变体Fc区的多肽)和具有增强或降低的C1q结合的多肽变体。
“抗体依赖性细胞吞噬作用”(ADCP)是指细胞介导的反应，其中表达FcγR的非特异的细胞毒性细胞识别靶细胞上的结合抗体，并随之引起靶细胞的吞噬。
本发明的抗体的氨基酸序列和核苷酸序列，以及Fc功能改造。
本发明首先采用重组SARS-CoV RBD蛋白来免疫小鼠，然后通过噬菌体抗体库筛选获得1株与SARS-CoV-2 RBD蛋白结合scFv抗体克隆。之后采用PCR方法将编码scFv抗体的重链和轻链可变区的核苷酸序列分别与编码小鼠IgG1重链恒定区和小鼠kappa轻链恒定区的核苷酸序列进行拼接，插入瞬转表达载体，进行培养表达。采用蛋白A纯化柱进行纯化获得高纯度鼠抗体。
采用经典的CDR移植方法进行鼠抗体的人源化改造 [8,9]。分别选择与鼠轻链和重链可 变区相似性均在50％以上，且轻链和重链可变区的框架区与待改造抗体轻链和重链可变区的框架区氨基酸序列相似性在50％以上的抗体做为人源化模板，将鼠抗体轻链或重链的3个CDR序列置换人源化模板中相应的CDR氨基酸序列中。通过比对IMGT人类抗体重轻链可变区种系基因数据库，选择出用于轻、重链可变区移植的人源模板。将鼠源抗体轻链和重链的3个CDR序列分别移植到相应的人源模板中。由于鼠源框架区的关键位点对于支撑CDR的活性至关重要，因此将关键点回复突变为鼠抗体的序列。分别将轻链/重链信号肽序列、回复突变的人源化抗体轻链/重链的可变区序列、人IgG4重链恒定区/人kappa轻链恒定区序列依次拼接，获得人源化抗体CoV2-HB27的氨基酸序列和核苷酸序列。
本发明进一步对于CoV2-HB27做Fc功能改造。分别是：1)基因工程改造的重链IgG1恒定区，得到降低Fc功能IgG1亚型人源化抗体CoV2-HB27-Fd6-IgG1；2)用Fut8基因敲除的哺乳动物细胞表达的去岩藻糖基化IgG1亚型CoV2-HB27-Ae0-IgG1抗体；和3)基因工程改造的重链IgG1恒定区，得到增强Fc功能IgG1亚型人源化抗体CoV2-HB27-Fe4-IgG1。
本发明的核酸
本发明还涉及编码本发明的抗体或其部分的核酸分子。这些核酸分子的一些示例序列见序列表。
本发明的核酸分子不限于本文公开的序列，还包括变体及与其对应的其他核酸形式，如mRNA,cDNA以及其变体。本发明中变体可以参照它们在杂交中的物理特性来描述。本领域技术人员会认识到利用核酸杂交技术，核酸可用于鉴别其互补物以及其等同物或同系物。还会认识到杂交可以以低于100％互补性发生。然而，考虑到条件的适当选择，杂交技术可用于基于DNA序列与特定探针的结构相关性来区分所述DNA序列。对于这类条件的指导参见Sambrook et al.,Molecular Cloning:A Laboratory Manual,2nd Ed.；Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989和Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Sedman,J.G.,Smith,J.A.,&Struhl,K.eds.(1995).Current Protocols in Molecular Biology.New York:John Wiley and Sons。
重组载体和表达
本发明还提供了包含本发明的一个或多个核苷酸序列的重组构建体。本发明的重组构建体可与载体一起使用，所述载体例如质粒、噬粒、噬菌体或病毒载体，编码本发明的抗体的核酸分子被插入所述载体中。
本文提供的抗体可通过在宿主细胞中重组表达编码轻链和重链或其部分的核苷酸序列来制备。为了以重组方法表达抗体，可用携带编码轻链和/或重链或其部分的核苷酸序列的一个或多个重组表达载体转染宿主细胞，以使得所述轻链和重链在所述宿主细胞中表达。标准重组DNA方法学被用于制备和/或获得编码重链和轻链的核酸、将这些核酸纳入重组表达载体中并且将所述载体引入至宿主细胞中，例如Sambrook,Fritsch and Maniatis(eds.),Molecular Cloning；A Laboratory Manual,Second Edition,Cold Spring Harbor,N.Y.,(1989)、Ausubel,F.M.et al.(eds.)Current Protocols in Molecular Biology,Greene Publishing Associates,(1989)和Boss et al.的美国专利No.4,816,397中记载的那些。
此外，可将编码所述重链和/或轻链的可变区的核苷酸序列转化为例如编码全长抗体链、Fab片段或scFv的核苷酸序列:例如可以将编码轻链可变区或重链可变区的DNA片段可操作地连接(以使得所述两个DNA片段编码的氨基酸序列都在框架中)至编码例如抗体恒定区或柔性接头的另一DNA片段。人重链和轻链恒定区的序列是本领域中已知的(参见，例如Kabat,E.A.,el al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)，包括这些区域的DNA片段可通过标准PCR扩增来获得。
为了表达所述抗体，可使用标准重组DNA表达方法(参见，例如Goeddel；Gene  Expression Technology.Methods in Enzymology 185,Academic Press,San Diego,Calif.(1990))。例如，可将编码所需抗体的核苷酸序列插入至表达载体中，随后将所述表达载体转染至合适的宿主细胞中。合适的宿主细胞为原核细胞和真核细胞。原核宿主细胞的实例为细菌，真核宿主细胞的实例为酵母、昆虫或哺乳动物细胞。应理解，包括选择调节序列的表达载体的设计受到多种因素的影响，例如宿主细胞的选择、所需的蛋白质的表达水平以及表达是组成型的还是可诱导型的。
本发明的抗体可通过公知方法从重组细胞培养物回收和纯化，所述公知方法包括但不限于，硫酸铵或乙醇沉淀、酸提取、蛋白A亲和层析、蛋白G亲和层析、阴离子或阳离子交换色谱法、磷酸纤维素色谱法、疏水相互作用色谱法、亲和色谱法、羟磷灰石色谱法以及凝集素色谱法。高效液相色谱法(“HPLC”)也可用于纯化。参见例如，Colligan,Current Protocols in Immunology,或Current Protocols in Protein Science,John Wiley&Sons,NY,N.Y.,(1997-2001)，例如第1、4、6、8、9、10章，各自以引用的方式全文纳入本文。
本发明的抗体包括天然纯化的产物、化学合成方法的产物和通过重组技术从原核及真核宿主产生的产物，所述真核宿主包括，例如酵母、高等植物、昆虫和哺乳动物细胞。本发明的抗体可以是糖基化的，或者可以是非糖基化的。这类方法记载于许多标准实验室手册中，例如上文的Sambrook，第17.37-17.42节；上文的Ausubel，第10、12、13、16、18和20章。
因此，本发明的实施方案还为包含所述载体或核酸分子的宿主细胞，其中所述宿主细胞可为高等真核宿主细胞例如哺乳动物及昆虫细胞、低等真核宿主细胞例如酵母细胞，并可为原核细胞例如细菌细胞。
本发明的抗体的特性和功能
ELISA测试表明，获得的鼠抗体CoV2-mhB27同瞬时表达SARS-CoV-2 S1蛋白HF-nCoV-SPIKE-8D3细胞有较好的结合，其结合EC 50为2.77μg/mL；能有效的竞争ACE2蛋白同RBD和S1蛋白的结合，竞争EC 50分别为44.0ng/mL和24.0ng/mL；可有效中和SARS-CoV-2的假病毒，并呈浓度依赖性，中和IC 50为8.7ng/mL。
人源化抗体CoV2-HB27与SARS-CoV-2 RBD、S1及ECD蛋白均具有较好的结合，结合呈“S”型曲线增长，结合EC 50分别为22.2ng/mL、183.3ng/mL和972.4ng/mL；可有效地抑制ACE2蛋白同SARS-CoV-2 S1蛋白的结合，抑制EC 50为178.6ng/mL。CoV2-HB27同SARS-CoV-2 RBD及S1蛋白均具有较高的亲和力。CoV2-HB27与SARS-CoV-2 RBD蛋白的亲和力为3.9E-11M，结合常数为3.7E+05 1/Ms，解离常数为1.4E-05 1/s，R 2为1.000；与SARS-CoV-2 S1蛋白的亲和力为3.7E-10M，结合常数为1.1E+06 1/Ms，解离常数为3.9E-04 1/s，R 2为0.997。CoV2-HB27可有效中和SARS-CoV-2的假病毒，并呈浓度依赖性，中和IC 50为5.9ng/mL。CoV2-HB27经单次静脉注射给予小鼠后，在体内的平均暴露量Cmax和AUClast分别为108.96μg/mL和9922.11h×μg/mL，平均半衰期t1/2为286.16h，清除率Cl为0.30mL/h/kg。
不同Fc功能形式的CoV2-HB27抗体中，去岩藻糖基化的Ae0-IgG1形式抗体和增强Fc功能的Fe4-IgG1形式抗体同CD16a的结合能力相似，强于IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同CD16a仅有极弱的结合。
不同Fc功能形式的CoV2-HB27抗体中，增强Fc功能的Fe4-IgG1形式抗体同CD32a和CD32b的结合能力最强，优于去岩藻糖基化的Ae0-IgG1形式抗体及IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同CD32a和CD32b几乎无结合。
不同Fc功能形式的CoV2-HB27抗体中，增强Fc功能的Fe4-IgG1形式抗体同CD64的结合能力最强，优于去岩藻糖基化的Ae0-IgG1形式抗体及IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同CD64仅有较弱的结合。
不同Fc功能形式的CoV2-HB27抗体中，增强Fc功能的Fe4-IgG1形式抗体同C1q 的结合能力最强，优于去岩藻糖基化的Ae0-IgG1形式抗体及IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同C1q仅有较弱的结合。
以Jurkat-NFAT/Luc2P-CD32A和Jurkat-NFAT/Luc2P-CD64为效应细胞时不同Fc功能形式的CoV2-HB27抗体中仅有增强Fc功能的Fe4-IgG1形式抗体在高浓度下可以介导较弱的ADCP作用，其余形式的抗体无ADCP作用。以Jurkat-NFAT/Luc2P-CD32B为效应细胞时，不同Fc功能形式的CoV2-HB27抗体均无ADCP作用。
不同Fc功能形式的CoV2-HB27抗体对表达SARS-CoV-2S蛋白的靶细胞均无CDC作用。
用途
本发明的抗体可用于治疗、预防或检测SARS-CoV-2病毒引起的疾病，如SARS-CoV-2病毒引起的急性呼吸道传染病。
药物组合物
可将本发明的抗体、抗原结合片段、改构抗体-药物分子、核酸、载体之一种或多种与至少一种其他化学剂制备成药物组合物，其包括上述活性成分和一种或多种药物可接受载体、稀释剂或赋形剂；任选地，还可以包含一种或多种其他治疗剂。
试剂盒
本发明还涉及药物包装和包含一个或多个容器的试剂盒，所述容器装有上文提到的本发明的药物组合物。与这类容器相关的可以是管理药物或生物制品的生产、使用或销售的政府机构所规定的形式的提示，其反映被所述产品的生产、使用或销售的机构批准用于人类给药。
制备和储存
本发明的药物组合物可以以本领域中已知的方式制备，例如通过常规的混合、溶解、造粒、研磨、乳化、包裹、包埋或冻干方法。
在已经制备包含配制于可接受的载体中的本发明化合物的药物组合物之后，可以将它们放置在适当的容器中并贴上标签用于治疗所标明的病症。这类标签会包括给药的量、频率和方法。
药物组合
上述包含本发明的抗体的药物组合物还与一种或多种其他治疗剂组合，其中所得组合不会引起不可接受的不利影响。
以下实施例用于示例性地说明本发明，而非对本发明进行限制。
实施例
实施例1：采用噬菌体抗体展示文库筛选结合SARS-CoV-2 RBD的鼠源抗体
1.1小鼠免疫
使用重组SARS-CoV-2-RBD-mFc蛋白(来源：北京义翘神州科技有限公司，Cat.40592-V05H，SEQ ID NO:1，下文同)免疫小鼠。具体方法为：将重组SARS-CoV-2-RBD-mFc蛋白与弗氏佐剂混合，使用混合物进行二次免疫，每次免疫剂量为50μg，皮下注射，免疫间隔为2周。第二次免疫后七天经眼眶内眦静脉丛采血。包被重组SARS-CoV-2-RBD-his蛋白(来源：北京义翘神州科技有限公司，Cat.40592-V08B)，采用ELISA方法检测小鼠免疫后的血清效价。第二次免疫血清滴度16000倍稀释达到2.228，10天之后处死小鼠，取小鼠的脾脏组织冻存于液氮中。
1.2噬菌体抗体库的构建
用TriPure Isolation Reagent试剂盒(来源：Roche，Cat.No.11 667 165 001)提取小鼠脾组织的RNA，用反转录试剂盒TriPure Isolation Reagent(来源：Invitrogen Cat.No.18080-051)进行反转录后获得cDNA。PCR扩增小鼠抗体轻链和重链可变区核苷酸序列后再采用重叠延伸拼接PCR法将编码小鼠抗体轻链和重链可变区序列的核苷酸序列拼接成编码scFv的核苷酸序列，轻重链可变区通过接头(linker)：
TCTAGTGGTGGCGGTGGTTCGGGCGGTGGTGGAGGTGGTAGTTCTAGATCTTCC(SEQ ID NO:2)
进行连接 [6]。通过限制性内切酶Sfi I(来源：Fermentas，下文同)酶切连接到噬菌体载体pComb3x(来源：北京义翘神州科技有限公司)中，电转化X-BLUE感受态(来源：Biomed，下文同)构建免疫小鼠的噬菌体展示scFv抗体库。
1.3结合SARS-CoV-2 RBD噬菌体的筛选
采用固相筛选法，筛选与SARS-CoV-2 RBD蛋白结合的噬菌体。第一轮筛选将浓度为10μg/mL的SARS-CoV-2-RBD-mFc蛋白包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，加入噬菌体库，37℃孵育2h。洗板去除未结合噬菌体，加入800μL Elution buffer(来源：神州细胞工程有限公司)孵育8min，然后每孔加入10μL 2M Tris buffer(来源：神州细胞工程有限公司，下文同)中和。将洗脱下的噬菌体浸染到X-BLUE菌种并加入辅助噬菌体表达，第二天收取表达后的噬菌体库。第二轮筛选方法同上。
从富集的文库中挑取单克隆噬菌体进行表达，用ELISA方法检测其与SARS-CoV-2 S及RBD蛋白的结合。将浓度为2μg/mL的SARS-CoV-2-S1-his蛋白(来源：北京义翘神州科技有限公司，下文同)、SARS-CoV-2-RBD-mFc蛋白及阴性对照CD155(D1)-mFc(来源：神州细胞工程有限公司)蛋白分别包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭2h后，洗板去除未结合蛋白，再加入10倍稀释的噬菌体单克隆孵育，洗板去除未结合的噬菌体，加入M13/HRP孵育后重复洗板。加入底物显色液进行显色，终止后酶标仪读取OD 450。以检测蛋白为横坐标，OD 450为纵坐标，利用GraphPad Prism软件分析并绘图。
结果如图1所示，从富集的文库中筛选获得1株与SARS-CoV-2 S1和RBD蛋白特异性结合的scFv克隆，CoV2-mB27，经测序获得scFv抗体的核苷酸序列(SEQ ID NO:3)。
1.4鼠源抗体的生产
PCR扩增CoV2-mB27scFv抗体的重链可变区核苷酸序列，通过In-fusion方法插入到带重链信号肽(SEQ ID NO:40)和人IgG1恒定区(SEQ ID NO:6)的经过ScaI+Nhe I(来源：Fermentas)酶切的pSE载体(来源：神州细胞工程有限公司，下文同)中获得人鼠嵌合抗体CoV2-mhB27重链(SEQ ID NO:36)的表达载体。PCR扩增CoV2-mB27scFv的轻链可变区核苷酸序列，通过In-fusion方法插入到带轻链信号肽(SEQ ID NO:41)和人kappa恒定区(SEQ ID NO:7)的经过ScaI+BsiWI(来源：Fermentas，下文同)酶切的pSE载体中获得人鼠嵌合CoV2-mhB27轻链(SEQ ID NO:37)的表达载体。
扩增可变区引物：
Figure PCTCN2021089748-appb-000001
用SCD4-4-TC2培养基(来源：北京义翘神州科技有限公司)传代293E细胞至200mL/瓶，起始接种密度为0.3～0.4×10 6cell/mL，37℃转速为175rpm的CO 2摇床中进行细胞培养。待细胞密度达到1.5～3×10 6cells/mL后，加入总量100μg按1:1混合的轻重链质粒DNA 和800μL的TF2转染试剂(来源：北京义翘神州科技有限公司)，在摇床中继续培养至第7天收料。培养液4000rpm离心25min，收集上清，加入1/5上清体积的stock buffer(来源：神州细胞工程有限公司)。用PBS将蛋白A层析柱(来源：神州细胞工程有限公司，下文同)平衡5～10倍柱体积，将过滤后的培养上清加入层析柱，再次平衡5～10倍柱体积后，用醋酸钠缓冲液(来源：神州细胞工程有限公司)洗脱样品。样品洗脱后用Tris buffer中和至中性备用。
实施例2：结合SARS-CoV-2 RBD鼠源抗体的功能检测
2.1鼠源抗体同SARS-CoV-2 S蛋白的结合
将不同浓度的SARS-CoV-2 S1蛋白(1000ng/mL、333.3ng/mL、111.1ng/mL、37.0ng/mL、12.4ng/mL、4.12ng/mL和1.37ng/mL)或SARS-CoV-2ECD蛋白(9000ng/mL、3000ng/mL、1000ng/mL、333.3ng/mL、111.1ng/mL、37.0ng/mL、12.4ng/mL和4.12ng/mL，来源：北京义翘神州科技有限公司，下文同)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，加入100μL 1μg/mL的鼠源抗体CoV2-mhB27孵育1h，之后洗板去除未结合抗体，加入0.25μg/mL Goat anti-human IgG Fc/HRP(来源：KPL公司，下文同)孵育后重复洗板，加入底物显色液进行显色，终止后检测OD 450。
结果如图2所示，CoV2-mhB27抗体与SARS-CoV-2 S1蛋白和ECD蛋白的结合均呈“S”型曲线增长，其结合EC 50分别为116.4ng/mL和306.6ng/mL。
本实施例进一步通过流式验证了鼠源抗体同瞬时表达SARS-CoV-2 S1蛋白HF-nCoV-SPIKE-8D3细胞的结合能力。取处于对数生长期HF-nCoV-SPIKE-8D3细胞(来源：神州细胞工程有限公司)置于流式管，5×10 5cell/管。加入不同浓度的鼠源抗体CoV2-mhB27(33.3μg/mL、16.7μg/mL、8.3μg/mL、4.2μg/mL、2.1μg/mL、1.04μg/mL和0.52μg/mL)，同时以H7N9-R1为阴性对照抗体(来源：神州细胞工程有限公司，下文同)。4℃混合孵育20min后PBS洗液清洗，离心去除未结合的抗体。加入FITC标记的Goat anti-Human IgG Fc二抗(来源：KPL公司)，4℃混合孵育20min后PBS重复清洗。200μLPBS重悬细胞，400目过滤后流式细胞仪上机检测。结果如图3所示，CoV2-mhB27抗体与HF-nCoV-SPIKE-8D3细胞有较好的结合，其结合EC 50为2.77μg/mL，阴性对照无结合。
2.2鼠源抗体竞争ACE2受体同SARS-CoV-2 RBD蛋白的结合
将浓度为1μg/mL的SARS-CoV-2 RBD或S1蛋白包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，加入100μL 0.08μg/mL的ACE2蛋白(来源：北京义翘神州科技有限公司，下文同)和不同浓度的CoV2-mhB27抗体(2500ng/mL、625ng/mL、156.3ng/mL、39.1ng/mL、9.8ng/mL和2.4ng/mL)共同孵育。洗板去除未结合抗体，加入0.5μg/mL C-his-R023/HRP(来源：神州细胞工程有限公司)孵育后重复洗板，最后加入底物显色液进行显色，终止后检测OD 450。抑制率PI％＝(OD 空白–OD 样品)/OD 空白×100％，其中OD 空白表示正常包被只加ACE2不加抗体组的OD值，OD 样品表示正常包被同时加ACE2和抗体的检测组OD值。
结果如图4所示，ACE2蛋白可结合包被的SARS-CoV-2的RBD和S1蛋白，CoV2-mhB27抗体均能有效的竞争ACE2蛋白同RBD和S1蛋白的结合，竞争EC 50分别为44.0ng/mL和24.0ng/mL。
2.3鼠源抗体鼠源抗体中和试验
使用Lennti-X 293(来源：Clontech)包装表达SARS-CoV-2 S全长蛋白的假病毒。将总量为62μg的PSD、pWPXL-Luc与pCMV3-SARS-CoV-2-S质粒(来源：神州细胞工 程有限公司)按照3:4:2的比例混合，加入72μL的Sinofection TF02(来源：北京义翘神州科技有限公司)转染试剂。混匀后室温放置10min，随后加入至Lenti-X 293细胞中。将细胞板置于37℃、5％CO 2的培养箱中培养6h后换液。继续培养48h后收取上清，用0.45μm的滤膜过滤去除细胞碎片，获得假病毒液，-80℃保存。
利用有限稀释法对病毒进行10倍梯度稀释，共设置10个病毒浓度，各6个复孔。96孔板中接种密度为3×10 4cell/mL过表达ACE2的293FT细胞(293FT-ACE2，来源：神州细胞工程有限公司，下文同)悬浮液，100μL/孔。每孔加入50μL梯度稀释的病毒，以细胞培养基作为阴性对照，混匀后置于37℃、5％CO 2的培养箱中静置培养24h。培养结束后加入5×passive lysis buffer(来源：Promega)，30μL/孔，混匀裂解细胞。取10μL/孔转入96孔白底板检测荧光信号，Reed-Muench法计算TCID 50值。
96孔细胞培养板中加入不同浓度的CoV2-mhB27抗体(2000ng/mL、571.4ng/mL、163.3ng/mL、46.6ng/mL、13.3ng/mL、3.8ng/mL、1.1ng/mL、0.31ng/mL和0.089ng/mL)，50μL/孔。每孔加入100TCID 50的假病毒，50μL/孔。以加病毒、不加抗体组作为阳性对照，不加病毒和抗体组为阴性对照。混匀后置于37℃、5％CO 2培养箱孵育1h。孵育完成后按照100μL/孔接入密度为3×10 4cell/mL的293FT-ACE2细胞悬浮液，混匀后置于37℃、5％CO 2培养箱中静置培养24h。培养结束后，加入5×passive lysis buffer(来源：Promega，下文同)，30μL/孔，混匀裂解细胞。取10μL/孔转入96孔白底板荧光信号值(RLU)，计算中和率。中和率％＝(阳性对照RLUs–样品RLUs)/(阳性对照RLUs–阴性对照RLUs)×100％。结果如图5所示，CoV2-mhB27可有效中和SARS-CoV-2的假病毒，并呈浓度依赖性，中和IC 50为8.7ng/mL。
实施例3：鼠抗体的人源化改造及生产
3.1鼠抗体轻链及重链的CDR确定
根据实施例1.3中测定的核苷酸序列推导出CoV2-mhB27中和抗体的重链可变区氨基酸序列(SEQ ID NO:8)和轻链可变区氨基酸序列(SEQ ID NO:9)。参考Kabat [7]以及IMGT编号方式确定CoV2-mhB27中和抗体轻链及重链各3个CDR的氨基酸序列，序列详见SEQ ID NO:10-15及表1。上述抗体的轻链及重链CDR在后续人源化步骤中直接移植到最终获得的人源化抗体CoV2-HB27中。
表1 鼠源抗体和人源化抗体的轻链及重链CDR序列
Figure PCTCN2021089748-appb-000002
3.2鼠抗体人源化CDR移植
采用经典的CDR移植方法进行鼠抗体的人源化改造 [8,9]。分别选择与鼠轻链和重链可变区相似性均在50％以上，且轻链和重链可变区的框架区与待改造抗体轻链和重链可变区的框架区氨基酸序列相似性在50％以上的抗体做为人源化模板，将鼠抗体轻链或重链的3个CDR序列置换人源化模板中相应的CDR氨基酸序列中。本实施例中通过比对IMGT人类抗体重轻链可变区种系基因数据库，选择出用于CoV2-mhB27的轻链可变区移植的人源模板为IGKV3D-11*02，该模板与CoV2-mhB27轻链的同源性为62.8％；重链可变区的人源模板为IGHV3-21*01，该模板与CoV2-mhB27重链的同源性为78.9％。
3.3人源化可变区序列框架区的回复突变
由于鼠源框架区的关键点对于维持CDR空间结构的稳定性具有至关重要的作用，因此需将关键点回复突变为鼠抗体的相应氨基酸。按照IMGT编号，将轻链的第42位回复突变为F，第71位回复突变为V，第83位回复突变为S,第88位回复突变为S,第103位回复突变为F；重链的第3位回复突变为K，第49位回复突变为R，第54位回复突变为A,第86位回复突变为T。经CDR人源化移植和框架区回复突变获得人源化抗体CoV2-HB27，其重链和轻链可变区氨基酸序列分别如SEQ ID NO:22/23所示；其含有信号肽的重链和轻链氨基酸序列分别如SEQ ID NO:18/19所示，分别包含依次连接的重链/轻链信号肽氨基酸序列(SEQ ID NO:20/21)；其人源化抗体重链/轻链的可变区氨基酸序列(SEQ ID NO:22/23)；其人源化抗体的恒定区为人IgG1重链恒定区/人kappa轻链恒定区序列(SEQ ID NO:32/33)。
3.4人源化抗体的生产
通过全基因合成的方法获得CoV2-HB27重链可变区核苷酸序列(SEQ ID NO:30)。通过In-fusion方法插入到带重链信号肽(SEQ ID NO:28)和重链IgG1恒定区(SEQ ID NO:32)的经ScaI+NheI(来源：Fermentas，下文同)酶切的pSE载体中获得CoV2-HB27重链(SEQ ID NO:26)表达载体。
通过全基因合成的方法分别获得CoV2-HB27轻链可变区(SEQ ID NO:31)核苷酸序列。通过In-fusion方法插入到带轻链信号肽(SEQ ID NO:29)和轻链kappa恒定区核苷酸序列(SEQ ID NO:33)的经ScaI+BsiWI(来源：Fermentas)酶切的pSE载体中获得CoV2-HB27轻链(SEQ ID NO:27)表达载体。
提质粒后转染HEK-293细胞(来源：Invitrogen，下文同)进行培养表达7天，采用蛋白A纯化柱纯化获得高纯度抗体。
全基因合成CoV2-HB27重链可变区引物：
F3(SEQ ID NO:56) GCTACCAGGGTGCTGAGTGAGGTGAAACTGGTGGAGTCTGGAGGAGGACTG
R3(SEQ ID NO:57) CAGGGAGCCTCCAGGCTTCACCAGTCCTCCTCC
F4(SEQ ID NO:58) CCTGGAGGCTCCCTGAGACTGTCCTGTGCTGCC
R4(SEQ ID NO:59) GTTGCTGAAGGTGAAGCCAGAGGCAGCACAGGA
F5(SEQ ID NO:60) TTCACCTTCAGCAACTATGGGATGAGTTGGGT
R5(SEQ ID NO:61) CTCTTGCCAGGAGCCTGTCTCACCCAACTCATC
F6(SEQ ID NO:62) GGCTCCTGGCAAGAGATTGGAGTGGGTGGCTG
R6(SEQ ID NO:63) AGGAGCCTCCAGAGGAAATCTCAGCCACCCACT
F7(SEQ ID NO:64) CCTCTGGAGGCTCCTACACCTACTACCCTGAC
R7(SEQ ID NO:65) GGTGAACCTGCCTGTCACTGTGTCAGGGTAGTA
F8(SEQ ID NO:66) ACAGGCAGGTTCACCATCAGCAGGGACAATGCC
R8(SEQ ID NO:67) TTGGAGGTAGAGGGTGTTCTTGGCATTGTCCCT
F9(SEQ ID NO:68) ACCCTCTACCTCCAAATGAACTCCCTGAGGGCT
R9(SEQ ID NO:69) GTAGTAGACTGCTGTGTCCTCAGCCCTCAGGGA
F10(SEQ ID NO:70) ACAGCAGTCTACTACTGTGCCAGGTTCAGATAT
R10(SEQ ID NO:71) CACTGTGCCTCCTCCTCCATCATATCTGAACCT
F11(SEQ ID NO:72) GGAGGAGGCACAGTGGACTACTGGGGACAAGGC
R11(SEQ ID NO:73) TGGGCCCTTGGTGCTTGCGCTGGACACTGTCACCAGGGTGCCTTGTCCCCA
拼接CoV2-HB27-IgG1重链引物：
Figure PCTCN2021089748-appb-000003
Figure PCTCN2021089748-appb-000004
全基因合成CoV2-HB27轻链可变区引物：
F12(SEQ ID NO:78) GCCACAGGAGTGCATAGTGAGATTGTGCTGACCCAGAGCCCTGCCACCCTG
R12(SEQ ID NO:79) CCTCTCTCCAGGGCTCAGGGACAGGGTGGCAGG
F13(SEQ ID NO:80) AGCCCTGGAGAGAGGGCTACCCTGTCCTGTAGG
R13(SEQ ID NO:81) GTTGTCCACAGACTCAGATGCCCTACAGGACAG
F14(SEQ ID NO:82) GAGTCTGTGGACAACTATGGCATCTCC
R14(SEQ ID NO:83) GGAACCAGTTCATAAAGGAGATGCCATA
F15(SEQ ID NO:84) TTATGAACTGGTTCCAACAGAAGCCTG
R15(SEQ ID NO:85) AGTCTTGGGGCTTGTCCAGGCTTCTGTT
F16(SEQ ID NO:86) ACAAGCCCCAAGACTGCTGATTTATGC
R16(SEQ ID NO:87) GCCCTGGTTGCTGGCAGCATAAATCAGC
F17(SEQ ID NO:88) GCCAGCAACCAGGGCTCTGGAGTGCCTGCCAGG
R17(SEQ ID NO:89) GCCAGAGCCAGAGCCAGAGAACCTGGCAGGCAC
F18(SEQ ID NO:90) GGCTCTGGCTCTGGCACAGACTTCTCCCTGACC
R18(SEQ ID NO:91) CTCAGGTTCCAAGGAGGAGATGGTCAGGGAGAA
F19(SEQ ID NO:92) TCCTTGGAACCTGAGGACTTTGCTGTCTACTTC
R19(SEQ ID NO:93) CACCTCCTTGCTCTGTTGACAGAAGTAGACAGC
F20(SEQ ID NO:94) CAGAGCAAGGAGGTGCCAAGGACCTTTGGACAA
R20(SEQ ID NO:95) TGGTGCAGCCACCGTACG CTTAATCTCCACCTTGGTGCCTTGTCCAAAGGT
实施例4：人源化抗体的抗原结合及中和能力检测
4.1人源化抗体同SARS-CoV-2蛋白的结合
将不同浓度的SARS-CoV-2 RBD蛋白(777.8ng/mL、259.3ng/mL、86.4ng/mL、28.8ng/mL、9.6ng/mL和3.2ng/mL)或S1蛋白(21000ng/mL、7000ng/mL、2333.3ng/mL、777.8ng/mL、259.3ng/mL、86.4ng/mL、28.8ng/mL、9.6ng/mL和3.2ng/mL)或ECD蛋白(21000ng/mL、7000ng/mL、2333.3ng/mL、777.8ng/mL、259.3ng/mL、86.4ng/mL、28.8ng/mL和9.6ng/mL)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1h后，加入100μL 1μg/mL的人源化抗体孵育1h，之后洗板去除未结合抗体，加入0.25μg/mL Goat anti-human IgG Fc/HRP孵育后重复洗板，加入底物显色液进行显色，终止后检测OD 450。
结果如图6所示，人源化抗体CoV2-HB27与SARS-CoV-2 RBD、S1及ECD蛋白均具有较好的结合，结合呈“S”型曲线增长，结合EC 50分别为22.2ng/mL、183.3ng/mL和972.4ng/mL。
4.2人源化抗体竞争ACE2受体同SARS-CoV-2 S1蛋白的结合
参照实施例2.2检测人源化抗体竞争ACE2受体同SARS-CoV-2 RBD蛋白结合的能力。结果如图7所示，人源化抗体CoV2-HB27可有效地抑制ACE2蛋白同SARS-CoV-2 S1蛋白的结合，抑制EC 50为178.6ng/mL。
4.3人源化抗体同SARS-CoV-2 S蛋白亲和力检测
采用生物分子相互作用分析系统(型号：OctetRED96e，厂家：Fortebio)测定人源化 抗体CoV2-HB27与生物素化的SARS-CoV-2 RBD和S1蛋白(来源：北京义翘神州科技有限公司)的结合亲和力。选用SA Sensor，平衡60s后加入2μg/mL的生物素化SARS-CoV-2 RBD和S1蛋白，再次平衡100s洗去未结合蛋白。加入不同浓度的人源化抗体(4.0μg/mL、2.0μg/mL、1.0μg/mL、0.5μg/mL、0.25μg/mL、0.13μg/mL和0.06μg/mL)300s，解离300s。用Data Analysis Octet软件对数据进行处理计算得到抗体亲和力(K D)、结合常数(kon)、解离常数(kdis)。
结果如表2所示，CoV2-HB27同SARS-CoV-2 RBD及S1蛋白均具有较高的亲和力。CoV2-HB27与SARS-CoV-2 RBD蛋白的亲和力为3.9E-11M，结合常数为3.7E+05 1/Ms，解离常数为1.4E-05 1/s，R 2为1.000；与SARS-CoV-2 S1蛋白的亲和力为3.7E-10M，结合常数为1.1E+06 1/Ms，解离常数为3.9E-04 1/s，R 2为0.997。具体动力学特征参数曲线见图8。
表2 CoV2-HB27同SARS-CoV-2 RBD及S1蛋白亲和力检测
Figure PCTCN2021089748-appb-000005
4.4人源化抗体中和SARS-CoV-2假病毒能力检测
参照实施例2.3评价人源化抗体CoV2-HB27(500ng/mL、200ng/mL、80ng/mL、32ng/mL、12.8ng/mL、5.12ng/mL、2.05ng/mL、0.82ng/mL和0.33ng/mL)在293FT-ACE2细胞中和100TCID50SARS-CoV-2假病毒的能力。结果如图9所示，CoV2-HB27可有效中和SARS-CoV-2的假病毒，并呈浓度依赖性，中和IC 50为5.9ng/mL。
实施例5：人源化抗体CoV2-HB27的药物质量和药物稳定性分析
5.1 CoV2-HB27的纯度和颗粒大小分析
应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS polyacrylamide gel electrophoresis，SDS-PAGE)、分子排阻高效液相色谱(size-exclusion high performance liquid chromatograph，SEC-HPLC)分析CoV2-HB27的纯度。SDS-PAGE具体操作步骤：(1)SDS-PAGE胶配制：3.9％浓缩胶，7.5％分离胶(非还原电泳)，13％分离胶(还原电泳)；(2)样品100℃煮沸2min，离心后上样8μg；(3)100V电泳15min后，调整电压为150V，继续电泳40min；(4)考马斯亮蓝染色后脱色，应用BandScan软件计算样品条带纯度。SEC-HPLC操作步骤为：(1)仪器：液相色谱系统(Agilent公司，型号：Agilent1260)，亲水硅胶高效分子排阻色谱柱(Tosoh公司，型号：TSK-GEL G3000SW XL(7.8×300mm，5μm))；(2)流动相：200mM NaH 2PO 4，100mM Arginine，pH6.5；(2)上样量为80μg；(3)检测波长280nM，分析时间为30min，流速为0.5mL/min，柱温度为25℃；(4)按面积归一化法计算各峰比例。
CoV2-HB27的还原SDS-PAGE、非还原SDS-PAGE的纯度分别为98.5％、83.9％；SEC-HPLC主峰比例为99.7％，聚集体比例为0.3％(表3)。两种检测结果均说明CoV2-HB27的纯度较高，除含有极少量聚集体外，无其他成分。
进一步应用动态光散射(dynamic light scattering，DLS)检测CoV2-HB27的颗粒大小和均一性，具体操作步骤：(1)仪器：动态光散射仪(Wyatt Technology公司，型号：DynaProNanoStar)；(2)上样量为50μL；(3)采集数据后，应用Dynamics 7.1.8软件分析数据。
CoV2-HB27的DLS检测结果显示，半径为5.8nM，百分分散度(％Pd)为9.8％，说明CoV2-HB27颗粒较小，为正常IgG1抗体颗粒大小，且均一性良好(表3)。
表3 CoV2-HB27的SEC-HPLC和DLS检测结果
Figure PCTCN2021089748-appb-000006
5.2 CoV2-HB27的热稳定性分析
应用差示扫描荧光法(differential scanning fluorimetry，DSF)检测CoV2-HB27的热稳定性。具体操作步骤：(1)仪器：Uncle系统(Unchained Labs公司，型号：UNCLE-0330)；(2)上样量为9μL；(3)设置检测参数：温度范围为25℃～95℃，升温速度0.3℃/min，每个温度下孵育30s；(4)应用UNcle Analysis软件分析数据，取UV266下内部荧光变化曲线的中点值为T m，取UV266/Blue473下静态光散射信号形成的聚集体变化曲线的聚合起始温度为T agg266和T agg473。
CoV2-HB27在组氨酸缓冲液(40mM Histidine，120mM NaCl，0.02％Tween80，pH6.0)中的热稳定性检测结果见表4，表现出了良好的热稳定性。
表4 CoV2-HB27的热稳定性检测结果
Figure PCTCN2021089748-appb-000007
5.3 CoV2-HB27的酸碱异构体分析
IgG1抗体因存在天冬酰胺(Asn)脱酰胺、赖氨酸(Lys)糖化、甲硫氨酸(Met)氧化等情况，故会出现电荷不均一的情况，表现出酸性和碱性异构体。我们分别应用阳离子交换高效液相色谱(cation exchange high performance liquid chromatograph，CEX-HPLC)、毛细管等电聚焦电泳(capillary isoelectric focusing，cIEF)分析CoV2-HB27的酸、碱性异构体水平。CEX-HPLC操作步骤：(1)仪器：液相色谱系统(Agilent公司，型号：Agilent1260)，阳离子交换色谱柱(Thermo公司，型号：ProPac TMWCX-10(4×250mm，5μM))；(2)流动相A：buffer A，pH 5.6(Thermo公司，货号：083273)；(3)流动相B：buffer B，pH 10.2(Thermo公司，货号：083275)；(4)上样量为80μg；(5)检测波长为280nM，分析时间为50min，流速为0.5mL/min，柱温度为25℃；(6)按面积归一化法计算各峰比例。cIEF操作步骤：(1)仪器：成像毛细管电泳仪(Proteinsimple公司，型号：iCE3)；(2)取10μL 5mg/ml样品与8μL Pharmalyte 3-10、70μL 1％methyl cellulose(MC)、2μL PI marker以及ddH 2O配制成分析液，总体积200μL；(3)将样品置于iCE3仪器中，1500V预聚焦1min，之后3000V聚集6min；(4)应用Chrom Perfect软件分析数据。
CoV2-HB27经CEX-HPLC检测后的酸性峰比例为9.7％，碱性峰比例为4.2％；经cIEF检测后的酸性峰比例为11.5％，碱性峰比例为4.5％(表5)。两种检测结果均表明，CoV2-HB27的酸性异构体、碱性异构体均处于较低的水平。
表5 CoV2-HB27的CEX-HPLC和cIEF检测结果
Figure PCTCN2021089748-appb-000008
5.4 CoV2-HB27的热加速稳定性分析
CoV2-HB27样品置于45℃中保存1周后，应用SDS-PAGE、SEC-HPLC分析样品的纯度，应用DLS分析样品颗粒大小的变化，具体操作步骤同5.1。
CoV2-HB27在45℃保存1周后，还原SDS-PAGE、非还原SDS-PAGE纯度均未下降；SEC-HPLC纯度下降1％，聚集体水平无变化，出现少量片段，说明CoV2-HB27在热加速后有较小的片段倾向，但热加速后的纯度仍较高。DLS检测结果显示，CoV2-HB27在热加速后的颗粒大小、％Pd均无变化，说明CoV2-HB27热加速后未出现聚集情况(表6)。总之，CoV2-HB27表现出了良好的热加速稳定性。
表6 CoV2-HB27的热加速稳定性检测结果
Figure PCTCN2021089748-appb-000009
5.5 CoV2-HB27的冻融稳定性分析
CoV2-HB27样品置于-80℃条件保存3h后转移至45℃条件解冻1h，如此进行五次反复冻融。应用SDS-PAGE、SEC-HPLC分析样品的纯度，应用DLS分析样品颗粒大小的变化，具体操作步骤同5.1。
CoV2-HB27经五次反复冻融后，SDS-PAGE、SEC-HPLC纯度无明显变化，聚集体、片段水平也无明显增加；DLS颗粒大小、％Pd也无明显增大(表7)。说明CoV2-HB27具有良好的冻融稳定性。
表7 CoV2-HB27的冻融稳定性检测结果
Figure PCTCN2021089748-appb-000010
5.6 CoV2-HB27的高浓度稳定性分析
将11.1mg/mL CoV2-HB27样品应用50kDa超滤管分别浓缩至26.0mg/mL、51.4mg/mL、85.5mg/mL、103.9mg/mL，应用SDS-PAGE、SEC-HPLC分析样品的纯度，应用DLS分析样品颗粒大小的变化，具体操作步骤同5.1。
CoV2-HB27浓度逐渐增加时，SDS-PAGE、SEC-HPLC纯度均无明显变化，聚集体、片段水平也无明显增加；但DLS颗粒半径随着浓度的增加而缓慢增大，最大半径为11.1nM(表8)。说明CoV2-HB27具有良好的高浓度稳定性。
表8 CoV2-HB27的高浓度稳定性检测结果
Figure PCTCN2021089748-appb-000011
Figure PCTCN2021089748-appb-000012
实施例6：CoV2-mhB27的表位分析
实施例2.2的结果表明，CoV2-mhB27可竞争ACE2受体同SARS-CoV-2 RBD和S1蛋白的结合。低温电镜结晶表明437-508号残基位点是SARS-CoV-2 RBD与ACE2结合所需的关键氨基酸残基 [10]。基于上述信息推测，在CoV2-mhB27结合RBD后，会与ACE2的结构构象形成空间位阻冲突。因此，本实施例选取了17个位于ACE2结合区及其附近的残基位点，将其突变为和原残基类型性质差异较大的其他残基类型，产生15个突变体，即E340R、T345Y、V367F、K378D、T385Y、N439R、N440Y、Y449A/N450、Y489R、T500Y、Y505E、A372Y、S375Y、D405R/R408和V503Y。
本实施例以SARS-CoV-2 RBD-His作为模板(序列来源：https://www.gisaid.org/)，使用PCR进行定点突变，并进行测序验证。瞬时转染表达突变型及野生型(WT)SARS-CoV-2 RBD蛋白并用ELISA检测CoV2-mhB27抗体同突变蛋白的结合能力。另外，为了检查抗体结合SARS-CoV-2 RBD突变体与WT相比的结合值差异的独特性，并排除潜在的突变体质量的影响，在研究中同时加入了一株非ACE2竞争的SARS-CoV中和抗体R007(来源：北京义翘神州科技有限公司)作为对照。
对于ELISA检测结果(图10)，先以WT SARS-CoV-2 RBD的ELISA读数作为标准，标准化各抗体检测结果。再针对某个特定的SARS-CoV-2 RBD突变体，以最高的抗体结合读数(设为100％)进一步标准化针对该突变体的ELISA结合信号。当mhB27针对特定突变体的ELISA结合信号相对于WT SARS-CoV-2 RBD下降到75％以下时，将该残基位点定义为显著结合表位。同样，若CoV2-mhB27针对特定突变体的ELISA结合信号下降到50％以下时，定义为高度显著表位。如图10所示，SARS-CoV-2 RBD中的N439和T500为CoV2-mhB27的高度显著表位。
实施例7：不同Fc功能形式人源化抗体CoV2-HB27的构建及生产
7.1降低Fc功能IgG1亚型人源化抗体CoV2-HB27的构建及生产
为降低抗体Fc片段介导的免疫功能，参照文献对IgG1亚型的恒定区进行核苷酸突变 [11]，得到基因工程改造的重链IgG1恒定区核苷酸序列(Fd6-IgG1，SEQ ID NO:44)。
通过PCR获得CoV2-HB27-Fd6-IgG1重链序列(SEQ ID NO:46)，其包含重链信号肽核苷酸序列(SEQ ID NO:28)，重链可变区核苷酸序列(SEQ ID NO:30)和Fd6-IgG1恒定区核苷酸序列(SEQ ID NO:44)。通过In-fusion方法插入经HindIII+XbaI酶切的pSE载体中获得含有CoV2-HB27-Fd6-IgG1重链(SEQ ID NO:46)的表达载体。
拼接CoV2-HB27-Fd6-IgG1重链引物：
Figure PCTCN2021089748-appb-000013
提取CoV2-HB27-Fd6-IgG1重链(SEQ ID NO:46)表达载体、CoV2-HB27轻链(SEQ  ID NO:27)表达载体质粒，转染HEK-293细胞进行培养表达7天，采用蛋白A纯化柱纯化获得降低Fc功能的CoV2-HB27-Fd6-IgG1抗体。
7.2去岩藻糖基化IgG1亚型人源化抗体CoV2-HB27的构建及生产
提取CoV2-HB27重链(SEQ ID NO:26)表达载体、CoV2-HB27轻链(SEQ ID NO:27)表达载体质粒，转染HEK-293(fut8基因敲除)细胞进行培养表达7天，采用蛋白A纯化柱纯化获得去岩藻糖基化IgG1亚型的CoV2-HB27-Ae0-IgG1抗体。
7.3增强Fc功能IgG1亚型人源化抗体CoV2-HB27的构建及生产
为增强抗体Fc片段介导的免疫功能，参照文献对IgG1亚型的恒定区进行核苷酸突变 [12,13]，得到基因工程改造的重链IgG1恒定区核苷酸序列(Fe4-IgG1，SEQ ID NO:48)。
通过PCR获得CoV2-HB27-Fe4-IgG1重链序列(SEQ ID NO:50)，其包含重链信号肽核苷酸序列(SEQ ID NO:28)，重链可变区核苷酸序列(SEQ ID NO:30)和Fe4-IgG1恒定区核苷酸序列(SEQ ID NO:48)。通过In-fusion方法插入经HindIII+XbaI酶切的pSE载体中获得CoV2-HB27-Fe4-IgG1重链(SEQ ID NO:50)表达载体。
拼接CoV2-HB27-Fe4-IgG1重链引物：
Figure PCTCN2021089748-appb-000014
提取CoV2-HB27-Fe4-IgG1重链(SEQ ID NO:50)表达载体、CoV2-HB27轻链(SEQ ID NO:27)表达载体质粒，转染HEK-293(fut8基因敲除)细胞进行培养表达7天，采用蛋白A纯化柱纯化获得增强Fc功能的CoV2-HB27-Fe4-IgG1抗体。
实施例8：不同形式人源化抗体CoV2-HB27的Fc功能
8.1不同Fc功能形式人源化抗体CoV2-HB27的CD16a结合功能
将浓度为10μg/mL的Avidin蛋白(来源：Thermo，下文同)包被于96孔板上，每孔100μL，2-8℃包被过夜。次日洗板，室温封闭1h后，加入100μL浓度为5μg/mL生物素标记的CD16a-AVI-His(V158)+BirA蛋白(来源：北京义翘神州科技有限公司)，室温孵育1h后洗板。分别加入100μL不同Fc功能形式的CoV2-HB27抗体，抗体加入浓度为40000ng/mL、10000ng/mL、2500ng/mL、625ng/mL、156.3ng/mL、39.1ng/mL、9.8ng/mL、2.44ng/mL、0.61ng/mL和0.15ng/mL。孵育1h后洗板去除未结合抗体，加入山羊抗人IgG F(ab)2/HRP(来源：Jackson ImmunoResearch公司，下文同)孵育后重复洗板，最后加入底物显色液进行显色，终止后酶标仪读取OD 450。
结果如图11所示，不同Fc功能形式的CoV2-HB27抗体中，去岩藻糖基化的Ae0-IgG1形式抗体和增强Fc功能的Fe4-IgG1形式抗体同CD16a的结合能力相似，强于IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同CD16a仅有极弱的结合。
8.2不同Fc功能形式人源化抗体CoV2-HB27的CD32结合功能
将浓度为10μg/mL的Avidin蛋白包被于96孔板上，每孔100μL，2-8℃包被过夜。次日洗板，室温封闭1h后，加入100μL浓度为5μg/mL的生物素标记的CD32a-AVI-His(R131)+BirA蛋白(来源：北京义翘神州科技有限公司)或CD32b-AVI-HIS+BirA(来源：北京义翘神州科技有限公司)蛋白，室温孵育1h后洗板。加入100μL不同Fc功能形式的CoV2-HB27抗体，抗体加入浓度为40000ng/mL、10000ng/mL、2500ng/mL、625ng/mL、156.3ng/mL、39.1ng/mL和9.77ng/mL。孵育1h后洗 板去除未结合抗体，加入山羊抗人IgG F(ab)2/HRP孵育后重复洗板，加入底物显色液进行显色，终止后酶标仪读取OD 450。
结果如图12所示，不同Fc功能形式的CoV2-HB27抗体中，增强Fc功能的Fe4-IgG1形式抗体同CD32a和CD32b的结合能力最强，优于去岩藻糖基化的Ae0-IgG1形式抗体及IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同CD32a和CD32b几乎无结合(图12A，12B)。
8.3不同Fc功能形式人源化抗体CoV2-HB27的CD64结合功能
将浓度为10μg/mL的Avidin蛋白包被于96孔板上，每孔100μL，2-8℃包被过夜。次日洗板，室温封闭1h后，加入100μL浓度为0.5μg/mL的生物素标记的CD64-AVI-His+BirA蛋白(来源：北京义翘神州科技有限公司)，室温孵育1h后洗板。加入100μL不同Fc功能形式的CoV2-HB27抗体，抗体加入浓度为2500ng/mL、625ng/mL、156.3ng/mL、39.1ng/mL、9.77ng/mL、2.44ng/mL、0.61ng/mL、0.15ng/mL、0.04ng/mL和0.01ng/mL。孵育1h后洗板去除未结合抗体，加入山羊抗人IgG F(ab)2/HRP孵育后重复洗板，最后加入底物显色液进行显色，终止后酶标仪读取OD 450。
结果如图13所示，不同Fc功能形式的CoV2-HB27抗体中，增强Fc功能的Fe4-IgG1形式抗体同CD64的结合能力最强，优于去岩藻糖基化的Ae0-IgG1形式抗体及IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同CD64仅有较弱的结合。
8.4不同Fc功能形式人源化抗体CoV2-HB27的C1q结合功能
将不同浓度的不同Fc功能形式的CoV2-HB27抗体分别包被于96孔板上，100μL/孔，4℃包被过夜，抗体包被浓度为40000ng/mL、10000ng/mL、2500ng/mL、625ng/mL、156.3ng/mL、39.1ng/mL和9.77ng/mL。次日洗板，室温封闭1h后，加入5μg/mL的C1q补体蛋白(来源：北京义翘神州科技有限公司)，100μg/孔，孵育1h。洗板去除未结合的蛋白，加入0.5μg/mL anti-His-MM02T/HRP(来源：北京义翘神州科技有限公司)孵育后重复洗板，最后加入底物显色液进行显色，终止后检测OD 450。
结果如图14所示，不同Fc功能形式的CoV2-HB27抗体中，增强Fc功能的Fe4-IgG1形式抗体同C1q的结合能力最强，优于去岩藻糖基化的Ae0-IgG1形式抗体及IgG1亚型的CoV2-HB27抗体；降低Fc功能的Fd6-IgG1形式抗体同C1q仅有较弱的结合。
8.5不同Fc功能形式人源化抗体CoV2-HB27介导的ADCC功能
以稳定表达SARS-CoV-2全长蛋白的HEK293FT单克隆细胞株(HEK293FT-SARS-CoV-2-S，来源：神州细胞工程有限公司，下文同)为靶细胞，以稳定转染CD16AV和NFAT-Luc2P的Jurkat细胞(Jurkat-NFAT/Luc2P-CD16AV)为效应细胞，利用报告基因法检测人源化抗体的ADCC功能。
在96孔板中按照50μL/孔接入密度为1×10 5cell/mL的靶细胞和等体积等密度的效应细胞。之后加入50μL不同Fc功能形式的CoV2-HB27抗体和H7N9-R1阴性对照抗体。CoV2-HB27-Ae0-IgG1、CoV2-HB27-He5-IgG1抗体加入浓度为6.25ng/mL、0.78ng/mL、0.097ng/mL、0.012ng/mL、0.0015ng/mL和0.00019ng/mL，其余抗体加入浓度为50ng/mL、6.25ng/mL、0.78ng/mL、0.097ng/mL、0.012ng/mL、0.0015ng/mL和0.00019ng/mL。混匀后37℃、5％CO 2培养箱中孵育6h。最后加入5×passive lysis buffer，30μL/孔，混匀裂解细胞。取10μL/孔细胞样品检测RLU值。利用GraphPad Prism软件分析并绘制量效曲线，横坐标为样品的浓度，纵坐标为RLU值。生物发光强度诱导倍数＝样品组RLU值/阴性对照组RLU值。
结果如图15所示，不同Fc功能形式的CoV2-HB27抗体中，增强Fc功能的Fe4-IgG1 形式抗体可介导最强的ADCC作用，优于去岩藻糖基化的Ae0-IgG1形式抗体及IgG1亚型抗体介导的ADCC作用；降低Fc功能的Fd6-IgG1形式抗体无ADCC作用。
8.6不同Fc功能形式人源化抗体CoV2-HB27介导的ADCP功能
以HEK293FT-SARS-CoV-2-S为靶细胞，以稳定转染CD32A，CD32B或CD64和NFAT-Luc2P的Jurkat细胞(Jurkat-NFAT/Luc2P-CD32A，Jurkat-NFAT/Luc2P-CD32B或Jurkat-NFAT/Luc2P-CD64)为效应细胞，利用报告基因法检测人源化抗体介导的ADCP功能。
在96孔板中按照50μL/孔接入密度为1×10 5cell/mL的靶细胞和等体积等密度的效应细胞。之后加入50μL不同Fc功能形式的CoV2-HB27抗体和H7N9-R1阴性对照抗体。以Jurkat-NFAT/Luc2P-CD32A，Jurkat-NFAT/Luc2P-CD32B为效应细胞时抗体加入浓度为50ng/mL、6.25ng/mL、0.78ng/mL、0.097ng/mL、0.012ng/mL、0.0015ng/mL、0.00019ng/mL、0.000024ng/mL和0.000003ng/mL；以Jurkat-NFAT/Luc2P-CD64为效应细胞时抗体加入浓度为6.25ng/mL、0.78ng/mL、0.097ng/mL、0.012ng/mL、0.0015ng/mL、0.00019ng/mL、0.000024ng/mL和0.000003ng/mL。混匀后37℃、5％CO 2培养箱中孵育6h。最后加入5×passive lysis buffer，30μL/孔，混匀裂解细胞。取10μL/孔细胞样品检测RLU值。利用GraphPad Prism软件分析并绘制量效曲线，横坐标为样品的浓度，纵坐标为RLU值。生物发光强度诱导倍数＝样品组RLU值/阴性对照组RLU值。
结果如图16所示，以Jurkat-NFAT/Luc2P-CD32A和Jurkat-NFAT/Luc2P-CD64为效应细胞时(图16A和图16C)，不同Fc功能形式的CoV2-HB27抗体中仅有增强Fc功能的Fe4-IgG1形式抗体在高浓度下可以介导较弱的ADCP作用，其余形式的抗体无ADCP作用。以Jurkat-NFAT/Luc2P-CD32B为效应细胞时(图16B)，不同Fc功能形式的CoV2-HB27抗体均无ADCP作用。
8.7不同Fc功能形式人源化抗体CoV2-HB27介导的CDC功能
以HEK293FT-SARS-CoV-2-S为靶细胞，利用WST-8法检测人源化抗体的CDC功能。
在96孔板中按照50μL/孔接入密度为2×10 6cell/mL的靶细胞。加入50μL的兔补体(来源：One lambda)和不同Fc功能形式的CoV2-HB27抗体并设置检测空白孔(无细胞)、阳性对照组(只接种细胞)对照和H7N9-R1阴性对照抗体组，抗体加入浓度为20ng/mL、4ng/mL、0.8ng/mL、0.16ng/mL、0.032ng/mL、0.0064ng/mL和0.00128ng/mL。混匀后37℃、5％CO 2培养箱中孵育2h。培养结束后加入WST-8显色液，10μL/孔。将96孔板置CO 2培养箱孵育，显色稳定后置酶标仪上于450nm、630nm处测定吸光度。以吸光度值(OD 450–OD 630)，并减去空白孔的读值来计算抗体的CDC的杀伤效应。杀伤率％＝(阳性对照OD值–样品OD值)/阳性对照OD值×100％。
结果如图17所示，不同Fc功能形式的CoV2-HB27抗体对表达SARS-CoV-2S蛋白的靶细胞均无CDC作用。
实施例9：不同Fc功能形式人源化抗体CoV2-HB27的小鼠药代动力学评价
9.1 IgG1型人源化抗体CoV2-HB27给予小鼠药代动力学试验
选用C57BL/6小鼠(共6只，雌雄各半，来源：北京维通利华实验动物技术有限公司)，单次尾静脉注射给予CoV2-HB27，给药剂量为5mg/kg，给药容积为10mL/kg。于给药前及给药后5min、30min、1h、3h、6h、10h、24h、32h、48h、72h、96h、120h、168h、240h、336h、504h和672h对所有小鼠进行眼眶采血，离心取血清。利用ELISA法检测血药浓度，并采用Phoenix-WinNonlin 8.1软件中非房室模型(NCA)计算药代动力学参数。
实验过程中所有小鼠均状态正常，药时曲线如图18所示，小鼠体内药物浓度随时间 不断变化，早期下降较快，但之后降低较缓慢，且无明显性别差异。单次药代动力学参数如表9所示，CoV2-HB27经单次静脉注射给予小鼠后，在体内的平均暴露量C max和AUC last分别为108.96μg/mL和9922.11h×μg/mL，平均半衰期t 1/2为286.16h，清除率Cl为0.30mL/h/kg。
表9 小鼠单次静脉注射CoV2-HB27的药代动力学参数(0～336h)
Figure PCTCN2021089748-appb-000015
9.2 CoV2-HB27-Fd6-IgG1给予小鼠药代动力学试验
选用C57BL/6小鼠(共6只，雌雄各半，来源：北京维通利华实验动物技术有限公司)，单次尾静脉注射给予CoV2-HB27-Fd6-IgG1抗体，给药剂量为5mg/kg，给药容积为10mL/kg。于给药前及给药后5min、30min、1h、3h、6h、10h、24h、32h、48h、72h、96h、120h168h、240h、336h、504h和672h对所有小鼠进行眼眶采血，离心取血清。利用ELISA法检测血药浓度，并采用Phoenix-WinNonlin 6.4软件中非房室模型(NCA)计算药代动力学参数。
实验过程中所有小鼠均状态正常，药时曲线如图19所示。小鼠体内药物浓度随时间不断降低，早期下降较快，但之后较长时间内变化较缓慢，且无明显性别差异。药代动力学参数如表10所示，CoV2-HB27-Fd6-IgG1经单次静脉注射给予小鼠后，在体内的平均暴露量C max和AUC last分别为115.04μg/mL和11159.82h×μg/mL，平均半衰期t 1/2为320.00h，清除率Cl为0.23mL/h/kg。
表10 小鼠单次静脉注射CoV2-HB27-Fd6-IgG1的药代动力学参数(0～336h)
Figure PCTCN2021089748-appb-000016
序列清单
Figure PCTCN2021089748-appb-000017
Figure PCTCN2021089748-appb-000018
Figure PCTCN2021089748-appb-000019
Figure PCTCN2021089748-appb-000020
Figure PCTCN2021089748-appb-000021
Figure PCTCN2021089748-appb-000022
Figure PCTCN2021089748-appb-000023
Figure PCTCN2021089748-appb-000024
Figure PCTCN2021089748-appb-000025
Figure PCTCN2021089748-appb-000026
Figure PCTCN2021089748-appb-000027
Figure PCTCN2021089748-appb-000028
参考文献
1.Zhao,S.,et al.,Preliminary estimation of the basic reproduction number of novel coronavirus(2019-nCoV)in China,from 2019 to 2020:A data-driven analysis in the early phase of the outbreak.International Journal of Infectious Diseases,2020.
2.Chen,N.,et al.,Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan,China:a descriptive study.The Lancet,2020.
3.Jiang,S.,et al.,A novel coronavirus(2019-nCoV)causing pneumonia-associated respiratory syndrome.Cellular&Molecular Immunology,2020:p.1-1.
4.Zhou,P.,et al.,Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.BioRxiv,2020.
5.Wan,Y.,et al.,Receptor recognition by novel coronavirus from Wuhan:An analysis based on decade-long structural studies of SARS.Journal of virology,2020.
6.Jones,S.T.and M.M.Bendig,Rapid PCR-cloning of full-length mouse immunoglobulin variable regions.Biotechnology(N Y),1991.9(6):p.579.
7.Kabat,E.A.,et al.,Sequences of proteins of immunological interest.1992:DIANE publishing.
8.Jones,P.T.,et al.,Replacing the complementarity-determining regions in a human antibody with those from a mouse.Nature,1986.321(6069):p.522.
9.Verhoeyen,M.and L.Riechmann,Engineering of antibodies.BioEssays,1988.8(2‐3):p.74-78.
10.Yan,R.,et al.,Structural basis for the recognition of the SARS-CoV-2by full-length human ACE2.Science,2020.
11.叶鑫,et al.,Cd47 antibody,antigen-binding fragment and medical use thereof.2018,Google Patents.
12.de Jong,R.N.,et al.,A novel platform for the potentiation of therapeutic antibodies based on antigen-dependent formation of IgG hexamers at the cell surface.PLoS biology,2016.14(1).
13.Parren,P.,et al.,Antibody variants and uses thereof.2014,Google Patents.

Claims (32)

  1. 一种分离的、封闭SARS-CoV-2刺突蛋白同ACE2受体的结合抗体或其抗原结合片段，其包含
    i)重链可变区，其重链CDR1、重链CDR2和重链CDR3域分别包含SEQ ID NO:13、14和15，和/或
    ii)轻链可变区，其轻链CDR1、轻链CDR2和轻链CDR3域分别包含SEQ ID NO:10、11和12。
  2. 如权利要求1所述的抗体或其抗原结合片段，其包含：
    i)重链可变区，其序列包含SEQ ID NO:22或与其具有至少85％、88％、90％、95％、98％或99％序列同一性；和/或
    ii)轻链可变区，其序列包含SEQ ID NO:23或与其具有至少85％、88％、90％、95％、98％或99％序列同一性。
  3. 如权利要求1-2之任一所述的抗体或其抗原结合片段，其为人源化抗体、嵌合或鼠源抗体。
  4. 如权利要求1-3之任一所述的抗体或其抗原结合片段，其抗体恒定区为IgG，IgM，IgA亚型，优选地，
    为IgG1、IgG2或IgG4亚型抗体；更优选地
    为因其Fc区的氨基酸序列和/或糖基化形式改变造成其与Fc受体和/或C1q补体结合功能改变的IgG1、IgG2或IgG4亚型抗体。
  5. 如权利要求1-4中任一所述的抗体或其抗原结合片段，其表位包含SARS-CoV-2病毒刺突蛋白的N439和/或T500。
  6. 如权利要求1-5之任一所述的抗体或其抗原结合片段，其中所述抗体进一步包含:
    i)重链恒定区，优选地，其序列包含SEQ ID NO:24或与其具有至少90％、92％、95％、98％或99％序列同一性；和/或
    ii)轻链恒定区,优选地，其序列包含SEQ ID NO:25或与其具有至少90％、92％、95％、98％或99％序列同一性。
  7. 权利要求1-6之任一所述的抗体或其抗原结合片段，或其亲和力优化抗体及其抗原结合片段，
    a)其与SARS-CoV-2 RBD的结合亲和力K D平均值为11.7E-11～1.3E-11M，优选5.9E-11～2.6E-11M,更优选为3.9E-11M；和/或
    b)其与SARS-CoV S1的结合亲和力K D平均值为11.1E-10～1.2E-10M，优选5.6E-10～2.5E-10M,更优选为3.7E-10M。
  8. 如权利要求7所述的抗体，其经单次静脉注射给予小鼠后，在体内的平均暴露量C max和AUC last分别为108.96μg/mL和9922.11h×μg/mL，平均半衰期t 1/2为286.16h，清除率Cl为0.30mL/h/kg。
  9. 如权利要求1-5所述的抗体，其中所述抗体进一步包含:
    i)重链恒定区，优选地，其序列包含SEQ ID NO:45或与其具有至少90％、92％、95％、98％ 或99％序列同一性；和/或
    ii)轻链恒定区,优选地，其序列包含SEQ ID NO:25或与其具有至少90％、92％、95％、98％或99％序列同一性。
  10. 如权利要求9的抗体，其
    同CD16a仅有极弱的结合；
    同CD32a和CD32b几乎无结合；
    同CD64仅有较弱的结合；
    同C1q仅有较弱的结合；
    无ADCC作用；
    无ADCP作用；
    无CDC作用。
  11. 如权利要求9或10所述的抗体，其经单次静脉注射给予小鼠后，在体内的平均暴露量C max和AUC last分别为115.04μg/mL和11159.82h×μg/mL，平均半衰期t 1/2为320.00h，清除率Cl为0.23mL/h/kg。
  12. 如权利要求6或9的抗体，其由fut8基因敲除的HEK-293表达。
  13. 如权利要求12的抗体，其
    同CD16a的结合能力强于IgG1亚型的CoV2-HB27抗体；
    显著介导对表达SARS-CoV-2S的ADCC作用。
  14. 如权利要求1-5所述的抗体，其中所述抗体进一步包含
    i)重链恒定区，优选地，其序列包含SEQ ID NO:49或与其具有至少90％、92％、95％、98％或99％序列同一性；和/或
    ii)轻链恒定区,优选地，其序列包含SEQ ID NO:25或与其具有至少90％、92％、95％、98％或99％序列同一性。
  15. 如权利要求14的抗体，其由fut8基因敲除的HEK-293表达。
  16. 权利要求14或15所述的抗体，其
    同CD16a的结合能力强于IgG1亚型的CoV2-HB27抗体；
    同CD32a和CD32b强于IgG1亚型的CoV2-HB27抗体；
    同CD64的结合能力强于IgG1亚型的CoV2-HB27抗体；
    同C1q的结合能力强于IgG1亚型的CoV2-HB27抗体；
    介导ADCC作用强于IgG1亚型的CoV2-HB27抗体；
    在高浓度下可以介导较弱的ADCP作用。
  17. 如权利要求1-16中任一项所述的抗体或其抗原结合片段，其为单克隆抗体。
  18. 如权利要求1-17任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段包含Fv、Fab、Fab′、Fab′-SH、F(ab′)2、Fd片段、Fd'片段、单链抗体分子或单域抗体；其中单链抗体分子优选包含scFv、di-scFv、tri-scFv、双体抗体或scFab。
  19. 一种改构抗体-药物分子，其包含如权利要求1-18任一项所述的抗体或其抗原结合片段 和与之共价或非共价连接的小分子或生物大分子，优选地通过接头连接。
  20. 一种核酸，其编码根据权利要求1-19任一项的抗体或其抗原结合片段，其为mRNA和/或DNA。
  21. 如权利要求20所述的核酸，其包含
    i)分别如SEQ ID NO:30所示的重链可变区核苷酸序列和/或如SEQ ID NO:31所示的轻链可变区核苷酸序列；和任选地
    ii)分别如SEQ ID NO:32、44、48所示重链恒定区核苷酸序列和/或如SEQ ID NO:7所示的轻链恒定区核苷酸序列；
    或i)和ii)的变体。
  22. 一种表达载体，其包含如权利要求20或21所述的核酸。
  23. 一种宿主细胞，其包含如权利要求20或21所述的核酸或如权利要求22所述的表达载体。
  24. 一种用于产生如权利要求1-18任一项所述的抗体或其抗原结合片段的方法，其包括在适合于抗体表达的条件下培养如权利要求23所述的宿主细胞，和从培养基中回收表达的抗体。
  25. 一种药物组合物，其包含
    如权利要求1-18任一项所述的抗体或其抗原结合片段或如权利要求19所述的改构抗体-药物分子或如权利要求20-21任一项所述的核酸或如权利要求22所述的表达载体；
    药学上可接受的载体；任选地
    一种或多种其他治疗剂，优选地，其他治疗剂选自抗病毒药物或炎性因子抑制剂、其他机制的小分子化学药；优选地，抗病毒药物选自包含不限于I型干扰素药物、抗体类、蛋白酶抑制剂类、RNA依赖性RNA聚合酶(RdRP)抑制剂类、靶向宿主的抗病毒类药物。
  26. 如权利要求1-18任一项所述的抗体或其抗原结合片段、如权利要求19所述的改构抗体-药物分子、如权利要求20-21任一项所述的核酸、如权利要求22所述的表达载体、如权利要求25所述的药物组合物，其用于预防和治疗SARS-CoV-2感染引起的疾病。
  27. 如权利要求1-18任一项所述的抗体或其抗原结合片段、如权利要求19所述的改构抗体-药物分子、如权利要求20-21任一项所述的核酸、如权利要求22所述的表达载体、如权利要求25所述的药物组合物在用于制备用于预防和治疗SARS-CoV-2感染引起的疾病的药物中的应用。
  28. 一种药物组合，其包含
    如权利要求1-18任一项所述的抗体或其抗原结合片段、如权利要求19所述的改构抗体-药物分子、如权利要求20-21任一项所述的核酸、如权利要求22所述的表达载体、如权利要求25所述的药物组合物；以及
    一种或多种另外的治疗剂。
  29. 一种试剂盒，其包含
    如权利要求1-18任一项所述的抗体或其抗原结合片段、如权利要求19所述的改构抗体- 药物分子、如权利要求20-21任一项所述的核酸、如权利要求22所述的表达载体、如权利要求25所述的药物组合物；优选地，
    还进一步包含给药的装置。
  30. 一种预防和治疗SARS-CoV-2感染引起的疾病的方法，其包含给予受治疗者
    如权利要求1-18任一项所述的抗体或其抗原结合片段、如权利要求19所述的改构抗体-药物分子、如权利要求20-21任一项所述的核酸、如权利要求22所述的表达载体、如权利要求25所述的药物组合物、如权利要求28所述的药物组合、或如权利要求29所述的试剂盒。
  31. 一种分离的、封闭SARS-CoV-2刺突蛋白同ACE2受体的结合抗体或其抗原结合片段，其结合表位包含SARS-CoV-2刺突蛋白的N439和T500。
  32. 一种SARS-CoV-2刺突蛋白的结合表位，其包含SARS-CoV-2刺突蛋白的N439和T500。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4145132A1 (en) * 2021-09-03 2023-03-08 ISAR Bioscience GmbH Methods and kit for determining the antibody status and t-cell immunity against sars-cov-2
WO2023031437A1 (en) * 2021-09-03 2023-03-09 Isar Bioscience Gmbh Methods and kit for determining the antibody status and t-cell immunity against sars-cov-2
WO2023092739A1 (zh) * 2021-11-26 2023-06-01 深圳先进技术研究院 抗sar-cov-2全人源单克隆抗体6g18及其制法与应用
CN116655786A (zh) * 2023-07-26 2023-08-29 中国人民解放军军事科学院军事医学研究院 广谱性抗SARS和SARS-CoV-2的抗体

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1884303A (zh) * 2005-06-20 2006-12-27 中国医学科学院基础医学研究所 SARS－CoV病毒结构蛋白的融合蛋白及其高量表达与纯化和用途
EP2193802A2 (en) * 2005-02-08 2010-06-09 The New York Blood Center, Inc. Neutralizing monoclonal antibodies against severe acute respiratory syndrome-associated coronavirus

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7491489B2 (en) * 2004-11-22 2009-02-17 The University Of Hong Knog Synthetic peptide targeting critical sites on the SARS-associated coronavirus spike protein responsible for viral infection and method of use thereof
CN110951756B (zh) * 2020-02-23 2020-08-04 广州恩宝生物医药科技有限公司 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
CN110974950B (zh) * 2020-03-05 2020-08-07 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2193802A2 (en) * 2005-02-08 2010-06-09 The New York Blood Center, Inc. Neutralizing monoclonal antibodies against severe acute respiratory syndrome-associated coronavirus
CN1884303A (zh) * 2005-06-20 2006-12-27 中国医学科学院基础医学研究所 SARS－CoV病毒结构蛋白的融合蛋白及其高量表达与纯化和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YAN RENHONG, ZHANG YUANYUAN, LI YANING, XIA LU, GUO YINGYING, ZHOU QIANG: "Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 367, no. 6485, 27 March 2020 (2020-03-27), US , pages 1444 - 1448, XP055798878, ISSN: 0036-8075, DOI: 10.1126/science.abb2762 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4145132A1 (en) * 2021-09-03 2023-03-08 ISAR Bioscience GmbH Methods and kit for determining the antibody status and t-cell immunity against sars-cov-2
WO2023031437A1 (en) * 2021-09-03 2023-03-09 Isar Bioscience Gmbh Methods and kit for determining the antibody status and t-cell immunity against sars-cov-2
WO2023092739A1 (zh) * 2021-11-26 2023-06-01 深圳先进技术研究院 抗sar-cov-2全人源单克隆抗体6g18及其制法与应用
CN116655786A (zh) * 2023-07-26 2023-08-29 中国人民解放军军事科学院军事医学研究院 广谱性抗SARS和SARS-CoV-2的抗体
CN116655786B (zh) * 2023-07-26 2023-10-27 中国人民解放军军事科学院军事医学研究院 广谱性抗SARS和SARS-CoV-2的抗体

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